Field of the InventionThis invention relates to an instrument, a method and labels forcarrying out assays using an electrochemical reaction on a conducting orsemi-conducting material in contact with an electrolyte but not inelectronic contact with an external electronic circuit.
The need for methods that provide rapid, sensitive, specific, preciseand low-cost detection of numerous analytes has led to the development ofa large number of differing assay methods and technologies. The desire andneed to improve these current methods still exists and numerouscompanies are working towards the development of improved means andthe exploration of new methods. The potential use of electrochemistry forthe development of assays which are rapid, sensitive, specific, precise andlow-cost has not escaped study due in part to this technique's ability todetect specific electronic properties directly caused by reactions at thesurface of an electrode electronically connected to an external electroniccircuit.
The use of particles, beads or other structures suspended in assaybuffers is now almost universal in immunoassay analyser systems available;examples are ELECSYS (Boehringer Mannheim), copalis technology(Sienna Biotech, Columbia MD), Luminex (Austin TX) with thefluorescence bead multiplex analysis system, IMMULITE (DiagProd.Corp), ACS:180 (Ciba Corning), Cobas Core (Roche), ACCESS(Sanofi), AIA-1200 DX (Tosoh), ACAplus (DuPont) and Immuno 1(Bayer). The reasons for using particles, beads or other structuressuspended in assay buffers is that they allow for rapid kinetics and alsofacilitate the manufacturing and automation of random access assaysystems. These also present some disadvantages related to keeping the beads in suspension, washing is not easy and problems can arise from theparticles, beads or other structures suspended in assay buffers interferingwith the detection systems.
When considering the detection and assay of analytes in gels, variousmethods are used to achieve a photographic-like analysis of the spatialdistribution of samples within the gel. Typically, this is achieved bystaining the gel directly with various agents to visualise the molecules inthe gel, for example commassie blue is used to detect proteins in gels. Theproteins bind the dye and are detected as blue bands. Ethidium bromide isused to detect DNA in gels as illumination with UV light allowsvisualisation of the fluorescence of the DNA-bound ethidium bromide. Insome cases the molecules are radiolabeled and are detected byautoradiography. The analytes may also be transferred to a membrane andanalysed after they have been bound. Examples of this are hybridizationssouthern, northern, and immunoblotting westerns. These methods aretypically time consuming and/or lack sensitivity.
Other methods for assaying analytes of interest are based on the useof filter wicking methods. These are used for various analytes such asglucose, cholesterol, drugs, antigens, antibodies and nucleic acids. In oneform, using antibodies, these are called immunochromatographic assays.These test formats using filter strip, wicking methods or lateral-flow offerthe beauty of simplicity. All that is necessary is to apply a sample, forexample blood, urine or saliva, and this results in the dried and labelledantibody dissolving and mixing with the sample and starting theantibody/analyte binding reaction. During this binding reaction thesample and labelled antibody are wicked (via capillary flow) along a stripwhich at a predetermined point contains an immobilised second antibodyagainst the analyte of interest. The result is that as the sample and labelledantibody are transported along the membrane or supporting phase, theanalyte binds to the antibody. This immune complex then at some point istransported in the liquid flow to the capture antibody which is immobilised at a given site within the filter or supporting phase. When theanalyte-labelled antibody complex interacts with the immobilised captureantibody it is captured by binding to an alternative epitope on the analyteof interest resulting in the immobilisation of the label which is attached tothe labelled antibody and complexed with the analyte. The fluid continuesto wick or flow past the immobilised antibody carrying any un-complexed(not bound to analyte of interest) labelled antibody away from theimmobilised capture antibody. At this point, the labelled antibody isdetected. These assays use a number of different labelling methods such asparticles and enzymes. In other systems based on these wicking or flowmethods, additional buffer and/or solutions can be added to give othertypes of signal and improve the binding reactions. In such a test, thesample is added and allowed to incubate. This is then followed by a wash,and the second binding reagent (labelled) is added followed by a wash. Thesubstrate is then added to detect the bound labelled reagent. These twoextremes of the filter wicking, lateral flow or immunochromatographicassay systems are given here to illustrate the potential range of conditionsand steps which can make up such rapid assay systems.
Electrochemical-based sensors for the detection of biomolecules havebeen developed into highly successful commercial products. This has beenexemplified by the measurement of glucose based on the detection ofelectrical changes that occur during an electrochemical reaction. Currentglucose detectors use an enzyme such as glucose oxidase to provide theselective recognition and generation of a detectable signal by an electrode.Some of these products, such as the system manufactured by BoehringerMannheim, use direct electrochemical oxidation of hydrogen peroxideproduced by the enzyme. In an alternative system, MediSense Inc. uses amodified ferrocene as a redox mediator between the enzyme and theelectrode. This removes problems due to oxygen concentrations and pH(M.G. Boutelle et al. J Mol. Rec. 1996. 9, 383-388). Electrochemicaltechnology has also been applied to the development of electrochemical methods based on the generation of light as a signal (Igen inc,Gaithersburg MD). In this system, a specific label, typically Ru (2,2-bipyridyl)32+,is used attached to a binding species through a spacer armand the capture of this label is detected by the generation of excited-stateRu (2,2-bipyridyl)32+ via a redox reaction at the surface of an electrodeconnected via an electronic circuit to a porentiostat. The voltage iscontrolled at the surface of the externally, electronically connectedelectrodes within the electrochemical cell to surface potentials less than 3volts due to the nature of the instrument and the electrochemistry of thissystem. This electrochemistry is conducted using a potentiostat to controlthe potential at a working electrode. These instruments for controllingelectrochemistry are designed to apply voltages of less than 10 volts andelectrochemical methods have thus been restricted to these low voltagesapplied to an externally, electronically connected electrode surface. In analternative electrochemical system which makes use of light as thedetectable signal, luminol and its derivatives can be subjected toelectrochemical excitation resulting in the emission of light. The use ofthe luminol system typically involves combination with a peroxidase-producingenzyme which generated hydrogen peroxide for the activationof the luminol light emission on electrochemical activation.
In summary, there are numerous examples of the use ofelectrochemistry for assaying biomolecules based on the detection ofspecific biomolecules directly via electrochemical methods or via the useof enzymes or other biocatalysis used to generate detectable species or viathe use of electrochemiluminescent labels as in the case of the rutheniumtrisbipyridyl label used by Igen inc, Gaithersburg MD (WO 86/02734).The essential element of all these methods has been the need to bring thereagents and/or binding species in close proximity to an electrode surfacewhich is in direct electronic contact via wires or equivalent electronconduction material to an external control electronic circuit. Theelectrode systems in these cases are typically made of a three-electrode system with working, counter and reference electrodes in contact with anelectrolyte that contains the sample. These three electrodes are then inelectronic contact outside the sample or electrolyte chamber via wire orequivalent electron conducting material to a potentiostat. Thisconfiguration of electrodes linked via a potentiostat allows precise controlof the voltages at the working electrode. In some cases, the electrodeconfiguration is made up of two electrodes and the counter and referenceelectrode are combined with some success. Thus, all electrochemical-basedassay systems have been designed using electrodes that are exposed to anelectrolyte containing the sample and connected by external contacts viawire or equivalent electron conducting material to a potentiostat. Theseelectrochemical assay systems make use of measurable electrochemistry,for example, by measuring electronic properties or light generated at thesurface of these directly controlled and connected electrodes. Thesesystems suffer from the problem of not being able to detect and analysethe presence of analytes in the bulk solution. If they are able to effectivelycapture the analytes in bulk solution, e.g. using beads, then these beadsneed to be captured or otherwise brought to the surface of an externally,electronically connected electrode. This presents a significant impedimentto the speed, sensitivity and simplicity of the assay and theinstrumentation, for example requiring a magnet (WO 96/15440, WO92/14139). In the case where the bead is brought to the surface of theelectrode, only a small portion of the assay sample is measurable (Williams1995 IVD Technology November 28-31). In cases where beads are notbrought into proximity of the working electrode, actual loss of signal hasbeen described when ECL labels are bound to the bead (WO 90/05301).
Systems involving electrochemical synthesis or production haveprovided examples of electrochemistry using electrodes consisting ofsuspended conducting particles. These have been described for theelectrolysis of sea water and the production of potassium permanganate(Fleischmann et al US4124453; Agladze et al US4269689; Bharucha et al US3941669). This field of electrochemistry has been called bipolarelectrochemistry as each conducting element serves as both anode andcathode ("Applications of Fluidized Beds in Electrochemistry" by P. LeGoff et al., Industrial & Engineering Chem., Vol. 61 No. 10, 1969, pp. 8-17.J.C. Bradley et al. Nature 1997, 389, 268-271). In these experiments,the electrolyte contains a suspension of conducting or semi-conductingparticles that are subjected to an electrical field. This results in thegeneration of anodic and cathodic faces on the conducting particles andresults in electrochemical reactions at the surface of the conductingparticles. These methods have not been used to generate a detectable signalor material which is related to a binding event or the presence of amolecule or analyte of interest for the purpose of determining the presenceor concentration of these molecules or analytes in a sample.
The present invention provides for the application of bipolarelectrochemistry in a new way for diagnostic applications and constitutesan improvement over those currently known. Various methods andexamples are disclosed.
Accordingly, it is an object of the invention to provide a method fordetecting molecules or analytes of interest using a conducting or semi-conductingmaterial - not in electronic connection with an externalelectronic circuit - as a bipolar electrode for electrochemically generatingdetectable signals or material. The level of detectable signal or materialbeing detected is related to the amount or presence of the molecules oranalytes of interest.
It is another and related object of the invention to describe anapparatus able to detect the signal or material electrochemically generatedby the conducting or semi-conducting material which is not in electronicconnection with an external electronic circuit.
It is another object of the invention to provide a new label forbinding assays which is a conducting or semi-conducting material acting asa bipolar electrode.
Accordingly, the invention is an improvement over the existing artin that it allows the electrochemical reaction to occur more effectively inthe bulk solution providing for more rapid assays of greater sensitivity. Itis a further improvement in that it provides a means for assaying multipleanalytes in an array without the need to provide multiple electrodecontacts or large extended electrode surfaces.
This invention includes an assay method for an analyte of interestusing an electrochemical process. The assay comprises the establishment ofcontact between the sample to be assayed for an analyte of interest and atleast one structure of conducting and/or semi-conducting material not inelectronic contact with an external electronic circuit; followed byapplying a voltage gradient across at least a portion of said at least onestructure of conducting and/or semi-conducting material not in electroniccontact with an external electronic circuit, said voltage gradient beingsuch that at least one electrochemical reaction takes place on at least oneof said at least one structure of conducting and/or semi-conductingmaterial not in electronic contact with an external electronic circuit; thendetecting and or quantitating said at least one electrochemical reactiondirectly and/or indirectly and determining the presence and/or quantityof said analyte of interest.
This invention also includes an apparatus for use in detecting and/orassaying an analyte of interest using electrochemistry, comprising; anelectrical power supply able to deliver a voltage; a cell able to accept asample containing structures of conducting and/or semi-conductingmaterial, wherein said cell contains at least two electrodes which areexternally and electronically connected to said electrical power supply andsaid cell is either an integral part of the apparatus or is a removable element, and a detector able to detect electrochemistry, said detectorselected from the following; film, photomultiplier tube, photodiode,phototransitor, CCD camera, electrochemical cell, wherein said detectordoes not or is not able to detect electrochemistry from said at least twoelectrodes.
This invention also includes a kit for carrying out an assaycomprising; a container with a binding species labelled with anelectrochemically-detectable species; a container with at least one structureof conducting and/or semi conducting material coupled with acomplementary binding species, wherein said at least one structure ofconducting and/or semi conducting material is capable of being stimulatedto carry out electrochemistry on application of an applied voltage to anelectrolyte in contact with said at least one structure of conducting and/orsemi conducting material when not in electronic contact with an externalelectronic control circuit.
This invention also includes an immunochromatographic devicecontaining;
- a binding species labelled with an electrochemically-detectablespecies,
- a binding species immobilised onto a solid phase
- and at least one structure of conducting and/or semi-conductingmaterial positioned such that electronic connection to an outside electricalcircuit can be avoided.
This invention also includes a method of activating a stabiliseddioxetane to emit light which comprises subjecting said dioxetane toelectrochemistry resulting in the destabilisation of said stabiliseddioxetane to induce its chemical decomposition resulting in light emission.
This invention also includes a method for generatingelectromagnetic radiation within a separations media which comprisesapplying a voltage to a separations media containing or constructed from structures of conducting and/or semi-conducting material such that saidseparations media is not rendered conductive and/or semi-conductive.
This invention relates to electrochemical-based assay systems foranalytes of interest such as molecules, ions, amine-containing molecules,antibodies, antigens, nucleic acids, receptors, ligands, hormones,biomolecules and any combination of binding species which, in operation,incorporate various structures of conducting or semi-conducting material.These various structures are not in electronic contact via a wire orequivalent electron conducting media to an external voltage supply. Thematerial of the subject invention is conductive or semi-conductive and actsas an electrode which has both an anodic and cathodic face under anapplied electric field. Thus, the material becomes bipolar with respect tothe developed electrical potential. The material is in electrical contact viaan electrolyte providing an ionic but not electronic contact to an externalelectronic circuit. The material is stimulated to carry out the desiredelectrochemical reactions by applying a voltage gradient across at least aportion of the electrolyte which is in contact with the conducting or semi-conductingmaterial. This results in the development of anodic andcathodic portions on the conducting or semi-conducting material surfacesuch that electrochemistry occurs on at least a portion of the anodic orcathodic portions of the conducting or semi-conducting material in contactwith the electrolyte. The result of the electrochemical reaction is toproduce a detectable signal (such as light) or to produce a detectablemolecular species (such as a chromophore or fluorophore orelectrochemically-reactive molecule) which allows the detection and/orquantitation of the analytes of interest. The detailed specific methods bywhich this can be brought about to allow detection and/or quantitation ofanalytes of interest are further described below.
It is well understood that electrical contact can be established byvarious methods and can be characterised by the mode of charge transfer.In one method for electrical contact, electrons move to achieve the chargetransfer. This form is typically known as electronic. This electronicmethod of electrical contact is based on electrons as carriers of charge andelectricity, as found in metals, conductors or semi-conductors. When anelectrolyte is used to create electrical contact and allow electricalconduction, no direct electronic conduction is involved. When anelectrolyte is used, the charge transferred during electrical conduction iscarried by ionic species (charged atoms and molecules). Thus, electronicconduction is not based on the movement of ionic species as chargecarriers. An electrolyte is an ionic conductor capable of forming anelectrical contact in all three states of matter: gases, solids and liquids.With electrolytes, charged molecular species move at the interface betweenan electrolyte and an electronic conductor or semi-conductor whereelectrochemistry can occur. This electrochemistry is dependent on theapplied voltage, chemicals (i.e. electrochemically-active compounds) in theelectrolyte and the nature of the electronic conductor or semi-conductorsurfaces.
Examples of materials which can be used in this invention to act aselectrodes to carry out electrochemistry are without limitation:conducting or semi-conducting materials, composites, mixtures, alloys,blends or other such obvious derivatives which combine many conducting,semi-conducting and non-conducting materials to produce materials whichin the final form are able to act as conductors or semi-conductors. Someexamples are Si, GaAs, Pt, Ag, Au, C, Zn, Si, Ge, Sn, Pb, Hg, Ti, V, Cr,Mn, Fe, Co, Ni, Cu, Mo, Ru, Rh, Pd, Cd, In, Sb, W, Re, Ta, Os, Ir, Bi,Al, tin oxide, indium/tin oxide (ITO), antimony/tin oxide, electronically-conductivepolymers such as polypyrole and polythiophene. Thesematerials may also be used as coatings e.g. carbon, gold, Pt, ITO and the conductive polymers. An example of a coated conductive material is ZelecECP which is an electronically-conductive powder (DuPont, DE). Zelec isavailable in two forms, one as a coating of antimony doped tin oxide on aninert core (silica, mica and titanium dioxide) or as solid particles ofantimony doped tin oxide. In an advantageous embodiment of theinvention C, Au, Pt, ITO and Ag are the most favoured conductingmaterials of interest in the invention as they present a number of idealproperties including cost, stability, availability and experience with thesematerials in a number of electrochemical systems. Also, these materialshave already been used to manufacture numerous structures, forms andcoatings. C is of particular interest because of the number of differentforms available and the number of differing electrochemical systems bothin electrochemical applications and also in electrochemical assay deviceswhich have made use of this material in its various conducting or semi-conductingforms. Carbon is also readily derivatized to yield numerousspecifically modified surfaces: such as in WO 97/33176 and WO 98/12539which are hereby incorporated by reference. In addition, many othermethods are known in the art for modifying carbon and other materials torender them useful for coupling. Examples are cited in the following; US5,667,667; P Allongue et al J Am Chem Soc 1997, 119, 201; C Sellitti et alMaterial Science and Eng.1990, A126, (1990) 235-244; C Kozlowski et al J.Chem. Soc., Faraday Trans. 1985, 81 2745-2756; B Barbier J. Electrochem.Soc. 1990, 137, 1757-1764; Au and Pt are also of special interest as thesematerials are used widely for electrochemistry, electronic fabrication andin immunoassay systems. Au and Pt also have added advantages over manyother materials in that they are relatively inert and are stable whenexposed to a wide range of chemical agents.
Examples of structures of conducting or semi-conducting materialssuitable for the subject invention are numerous. These structures can takenumerous shapes and forms, such as beads, particles, spheres, spheroids, cylinders, cones, plates, coatings, fibres, fibrils, nanotubes, nanoparticles,colloids, splinters, filings, springs, coils, amorphous particles, amorphousplates or coatings, and as a conductive coating may take the form of twodimensional shapes of squares, triangles, rectangles, circles, pentagons,hexagons, stars etc. In an advantageous embodiment, the structures ofmost interest will be particles that are spherical or spheres or similar, alsofibres, fibrils and nanotubes for the coatings. These may be in the form ofconductive coatings on the surface of 3 dimensional structures, but canalso be in the form of 2 dimensional shapes such as those produced by theevaporation of gold onto a surface which is masked to produce a desiredshape on a surface. Other examples of coating methods are withoutlimitations, e.g. sputtering, screen printing, plating, polymerisation,stamping and painting. Many materials can be coated onto surfaces of flator shaped structures thus producing a desired conducting or semi-conductingsurface to form a structure for the purposes of the invention.
In the cases of evaporated gold, sputtered carbon shapes orpyrolysed carbon on a surface, the potential exists to generate an array ofsuch gold or carbon elements where each may function as separate bipolarelectrodes as described for this invention. Other coating methods, asillustrated above, could also be used in this way to generate a series ofpotential bipolar electrodes. The present invention using arrays ofpatterned conducting elements does not need to be in electronic contactwith an outside circuit. As a result, the invention solves a probleminherent to the application of multiple electrodes or multiarray electrodesused in the development of electrochemical assay systems for multiplesamples and multiple analyte detection systems.
In the case of particles or beads, it is understood that particles orbeads from cm to nm in size have been used in immunoassay applicationsranging from tube-based assays to filter wicking assay systems. Weanticipate the use of particles in these ranges as structures of the subjectinvention.
It is also anticipated that the structures of conducting and/or semi-conductingmaterial may also be incorporated into composite materials bymixing with non-conducting and/or conducting and/or semi-conductingmaterials. By way of an example, filters may be constructed by theinclusion of structures of conducting and/or semi-conducting materialduring manufacture to generate a non-conducting filter material whichcontains dispersed structures of conducting and/or semi-conductingmaterials. These composite filters are valuable in the development of theimmunochromatography methods of the invention. When wetted withelectrolyte, these composite filters can be activated to carry outelectrochemistry through the bulk of the filter at the sites of the dispersedstructures of conducting and/or semi-conducting material. In other formsof composites the concentration of the conduction and/or semi-conductingmaterial is such that the composite is itself conducting and/or semi-conducting.The composites of this type are then equivalent to theconducting and/or semi-conducting materials of the subject invention.These composites can then be used to form the structures of the inventionas described above forming for example fibres, particles, beads etc. Othertypes of composite containing conductive and semi-conductive structuresof the subject invention are gels and sols and hydrogels. In thesecomposites with gels, sols and hydrogels, the conductive material isdispersed or suspended in such a way that the conductive or semi-conductivestructures do not render the composite electronicallyconducting. In addition, the amount of conductive or semi-conductivestructures is such that the final form of the composite is visible to variouselectromagnetic radiation and photon detection means. Also, suchcomposites are amenable to the use of conventional electrochemicaldetection methods using electrochemical cells externally connected to aelectronic circuit.
Some examples of materials which are readily available and illustratesome of the forms contemplated in this invention are: Aluminium powder, 20 micron; Beryllium powder, 10-20 micron; glassy carbon sphericalpowder in various sizes from 0.2 µm to 2,000 µm; graphite powders;graphite fibre 8 µm in diameter and 6-25 mm long; gold powder, spherical,2-8 µm; gold flake, 2-15 µm; nickel powders, 2-7 micron, various forms;palladium powder, various forms from 0.25-7.5 micron; platinum powder,various forms, 0.2-3 micron (Alfa Aesar, Ward Hill, MA); carbonnanotubes or fibrils (Materials and Electrochemical Research Corp,Tucson, AZ); Zelec ECP (DuPont, DE).
In addition, the fabrication of the structures from the materials ofthe invention is highly developed allowing a wide range of shapes andstructures as outlined above. For example, so-called nanowires which canbe up to 55 µm long and about 250 nm in diameter have been made(Huber, CA, Science 1994, 263, p800). It is contemplated that nanowires ofvarious sizes and material can be used advantageously in the presentinvention as structures of the subject invention. It is also contemplatedthat structures of the subject invention, as described above, may be coatedto improve the electrochemistry or reduce interferences, for instance withgels, polymer layers or grafts and solid electrolytes layers which areknown in the art.
Detectable electrochemical reactions consist of electrochemistry on aspecies (a so-called electrochemically-detectable species) which gives rise toa directly detectable species such as photons of electromagnetic radiationor indirectly via production of a subsequently-detectable species ormolecule such as a chemiluminescent, fluorescent or coloured molecule ora molecule with an altered absorption spectrum, and electrochemicallyactive species.
Numerous examples of detectable electrochemical reactions exist andproduce electromagnetic radiation, electrochemically active molecules,chromogenic molecules and fluorogenic molecules. Advantageously, thedetectable reaction is directly coupled (i.e. comprising typically no more than four to five reaction steps) to the electrochemical reaction at theelectrode surface but may also be indirectly coupled to the reactions at theelectrode surface where numerous reactions linked to an initial electrodereaction result in the production of a detectable signal, species and/ormolecule.
The electrochemical reactions that produce electromagneticradiation, such as ECL, chemiluminescence, and electroluminescence, areeasy to detect as they require simply a means to detect the electromagneticradiation produced. In the case of ECL and chemiluminescence, theelectromagnetic radiation is produced from electrochemical reactionseither directly or by coupling to other electrochemically-producedreactants. Various forms of ECL are considered in this invention, forexample, ECL from Luminol with hydrogen peroxide, ECL from metalchelates and organic molecules which can occur in the presence of co-reactantssuch as oxalate (D Ege et al, 1984, Anal Chem 56, 2413-2417) andnumerous amines as described in WO 97/33176 and EP 0441894B1 whichare incorporated by reference in their entirety. Also, the ECL fromLuminol with hydroperoxides, e.g. in the measurement of lipidhydroperoxides (A. Zamburlino, Biochim Biophys Acta (1995) 1256, 233-240).ECL may also be generated from a number of metal chelates andorganic molecules where use is made of the electrochemically-generatedoxidised and reduced species (generated at the anode(s) and cathode(s)) toproduce the excited state for light emission. Light emission may also beactivated electrochemically from other chemiluminescent molecules inaddition to luminol such as acridinium ester and advantageously fromstabilised dioxetanes such as those sold by Tropix (Waltham, MA). In thecase of the stabilised dioxetanes, these have typically been stabilised by theuse of substituent groups which can be removed enzymatically todestabilise the dioxetane and thus produce light. In the subject invention,the dioxetanes may be stabilised by groups which are or can beelectrochemically removable providing for a wider range of potential chemistries; examples of such groups are alcohols, acids, and glycosides(VG Mairanovsky, 1976, Angew. Chem. Int. Ed. Engl. 15, 281-292).
It is also contemplated that fluorescence would be used as adetectable signal or label. Fluorescence could be used either via thesynthesis or the activation of a fluorescent species. For example, the localpH changes brought about in the proximity of a micro electrode can besensed by the fluorescein activation caused by the pH change in the localenvironment of the electrochemical reaction (BB Ratcliff et al 1996, AnalChem 68, 2010-214). An electrogenerated fluorescent species may also bedetected as demonstrated by McLeod et al, (The Analyst, 1982, 107, 1-11).
In another example, the electrochemical reaction, which isdetectable, allows the detection of the conducting and/or semi-conductingstructure. In this example, the conducting and/or semi-conductingstructure becomes the detectable species and can be used to detect theanalyte of interest. This property has been described for Al which canemit light on application of a voltage at anodic and cathodic surfaces(Haapakka K et al, 1988, Anal Chim Acta 207, 195-210).
A conducting and/or semi conducting structure may also be coupledto a binding species to form the electrochemically-detectable species.When bound or captured, this could subsequently be detected usingelectrolytes with ECL and CL species such as Ru(bipy), luminol. Thecapture of a structure of the subject invention would allow forconsiderable enhancement of the signal as a single binding event wouldprovide a bipolar electrode which could generate more light in an ECLreaction than from the capture of a few ECL active molecules. Theconducting and/or semi conducting structure may be made from or coatedwith electroluminescent material and in this form no additional chemistrywould be needed to support the generation of light from structures in theelectrolyte when a suitable voltage is applied.
The ECL labels of the subject invention include numerous examplesof both organic compounds and metal ion chelates. Well known examplesare Ru tribipy, Ru tris phen, Os tribipy, and numerous other metalchelates described in the literature, and luminol. Numerous examples arecovered in the following publications: US5453356, US5591581, US5238808,US5221605, WO 97/33176, WO 96/28558 which are incorporated byreference in their entirety. Also, Al structures are known to luminescewhen coupled to an electrochemical reaction in an electrolyte (HaapakkaK et al, 1988, Anal Chim Acta 207, 195-210).
The electrolyte of the present invention typically contains ionicspecies in solutions, solids and gases to allow electrical conducting. Acharge passing through an electrolyte is transferred via movement of theionic species in the bulk solution, solid or gas. In addition to these basicelements, in many cases the ionic species is also involved in the control ofthe ionic strength and/or the pH of the electrolyte such that it is optimalfor the assay of the analyte of interest and/or the electrochemistry.Additionally, it is common to find within the electrolyte the addition ofcoreactants used to enhance or support the electrochemistry of thedetection system. An example of an electrolyte that can be used for ECLwith Ru (2,2-bipyridyl)32+ is phosphate buffer used as electrolyte and pHcontrol, combined with tripropylamine as an amine coreactant to supportthe ECL electrochemistry.
Numerous examples of buffers exist, for example borate, phosphate,tris, HEPES, MES, MOPS, PIPES, acetate, triethylamine, tripropylamine,bicarbonate with the appropriate counter ion. In some cases, the buffermay also be a coreactant e.g. amine-containing buffers can act ascoreactants in Ru (2,2-bipyridyl)32+ -based ECL.
The ionic strength of the medium can be controlled typically byadding salts, but may be established by controlling the concentrations ofthe buffer and/or the coreactants. Examples of salts which may be used to control the ionic strength are numerous. Examples are salts of K, Na, Ca,ammonium, guanidinium.
Typically, electrolytes of the subject invention are based on watersolutions, but organic solvents may be used and in some casesadvantageously during the electrochemical reaction. It will be understoodthat mixed solvents can also be used advantageously. For example,formamide is used advantageously in DNA probe applications.
Other components may also be added to improve assay conditions.Typical examples of components used to improve immunoassays aredetergents, other proteins and lipid components. These other componentsare known in the art and are typically optimised for various assays underthe various conditions of differing assay systems. The optimisation ofthese components is well known in the art and forms an integral part ofassay optimisations. The use of other components to enhance variousaspects of nucleic acid probes is also known in the art.
Basically, the instruments integrate a luminometer, a voltage controlcircuit and a cell with two externally electronically connected electrodes.Optionally, the apparatus may include a pump or other fluid-handlingcomponents and a flow-through cell with externally electronicallyconnected electrodes.
The instruments of the subject invention advantageously have a cellwhich contains at least one electrolyte. This cell contains at least twoelectrodes which are in electronic contact with an external electricalcircuit which is able to apply a voltage. These two electrodes are incontact within the cell via the electrolyte within the cell. It is possible todesign cells of various shapes and configurations such as flow-throughcells, single-chamber cells which allow the introduction of electrolyte atdesired times. It is also possible that the cell and electrodes are a singlemodule which can be inserted into the instrument to make contact. Theexternally electronically connected electrodes may be isolated from a central zone or at least from each other by a membrane, gel or othermaterial which is semi permeable to ions and thus maintains contactbetween the two externally electronically connected electrodes via theelectrolyte. This use of a semi permeable membrane may be valuable forisolating the sample from the externally electronically connectedelectrodes and from the electrolyte in which they are immersed. It mayalso be valuable to prevent both the detectors and the sample from beingaffected by the electrochemistry occurring on the externally electronicallyconnected electrodes.
Ideally, the various instruments contemplated would be controlledby a microprocessor either directly by incorporation of a microprocessorinto the instrument and/or by an external microprocessor such as acomputer. Software for controlling such interfaces is well known to thosein the art and may also be written for custom applications to make use ofinternal and external microprocessor control e.g. Labview from NationalInstruments (Austin Tx) www.natinst.com. In addition it will beunderstood that simple electronic circuits can be constructed to controlthe application of voltage to the cell. Thus, the control of such aninstrument can be achieved at various levels from simple mechanicalsystems to microprocessor controlled systems.
An example of an instrument of the subject invention contains a cellwhich is able to contain an electrolyte of interest for the detection and/orquantitation of an analyte(s) of interest which further consists of threezones or functionally distinct areas. Two of the zones flank a central zoneand contain externally (to the cell) electronically connected electrodes.These two externally electronically connected electrodes are used to applyvoltages to the central zone of the cell. The two externally electronicallyconnected electrodes are externally connected to a controlled voltagesource. Typically, this controlled voltage source is under the control of acomputer but may be controlled by other means such as a simple electricalcircuit or mechanical means for triggering the application of the applied voltage. The controlled voltage applied to at least the central zone isinitiated by an operator or by determining the presence of, or placing, thesample electrolytes in the central zone of the cell. The sample electrolytecontains the conducting and/or semi-conducting structures of the subjectinvention along with the compound(s) which are able electrochemically toproduce a detectable species allowing detection of analytes. The twoexternally electronically connected electrodes electronically connected tothe controlled voltage source are thus able to apply a desired voltagegradient to the central zone of the cell controlled by electronic meansincluding a simple electronic circuit and/or microprocessor. Theapplication of this desired voltage to the central zone of the cell allows theconducting and/or semi-conducting structures of the subject invention tocarry out electrochemistry. This desired electrochemistry results in theformation directly or indirectly of a detectable species which whendetected allows the detection and/or quantitation of the analyte ofinterest.
The central zone of the cell is fashioned in such a way that it readilyallows the detection of the detectable species generated by theelectrochemistry. In this regard, the central zone will contain at least onewindow. This window will permit the detection of electromagneticradiation from the central zone. In an alternative embodiment, thewindow is present and additional externally and electronically connectedelectrodes are introduced through the open window into the central zone,or into a zone in a flowpath from the central zone, to allowelectrochemical detection of the detectable species using conventional andunderstood electrode configurations in combination with conventionalelectrochemical methods such as amperometric or potentiometric methods.Examples of these are glucose and oxygen sensors and oxygen electrodes.
In the case of ECL and chemiluminescence, the window will permitphoton detection by a means of choice to detect and/or quantitate thephotons from the central zone.
In the case of a chromogenic detectable species, the central zonewould allow a beam of photons to pass through the sample in the centralzone and be analysed and/or detected and/or quantitated. The analysis ofthis beam of photons after passing through the sample in the central zonewould determine whether the intensity of the photon beam had changedand/or if the spectral properties of the photon beam had been changed. Inan advantageous embodiment, the beam of photons would have awavelength selected to interact optimally with the detectable chromogenicspecies of interest.
In the case of a fluorogenic detectable species, the central zonewould allow a beam of photons to pass through the sample and be analysedand/or detected and/or quantitated. The analysis of this beam of photons,after passing through the sample in the central zone, would determine thenumber and/or intensity of photons emitted from the sample in thecentral zone and would determine whether the spectrum had changed withrespect to the incident beam of photons. In an advantageous embodiment,the beam of photons would have a wavelength selected to interactoptimally with the detectable fluorogenic species of interest and thedetector would be optimised to detect and/or quantitate the emittedphotons from the detectable fluorogenic species. The use of time-resolvedfluorescence is also a contemplated detection means. The use offluorescence and/or other energy transfers is also contemplated as a meansof signal enhancement and discrimination, as used in other assay systemsknown in the art. Other detection means are also contemplated by thisassay system and instrument, such as electrochemical means for theproducts of the electrochemical reactions at the surface of the structures ofthe subject invention.
Various cells for the instrument of the subject invention arecontemplated and it is understood that numerous potential examples arepossible. Examples are described in detail in the section "Examples".
In the case for an instrument able to analyse clinical samples, a flow-throughcell with the three zones described above would be advantageous.
When using the subject invention with rapidimmunochromatography-based assays, a cell would be a section of such adevice. This section could be removable and is potentially a disposableelement.
In a multiwell plate, the structures of the invention could be coatedonto the surface of the well in a microtitre plate, typically on the bottomor portions of this part of the well, either as a single contiguousconducting or semi-conducting structure or advantageously as multipleelements in the size range of 1 µm to 1 mm as measured between the edgesof the individual structures in the plane between the externallyelectronically connected electrodes. The wells in this multiwell formatcould have the externally electronically connected electrodes within thewell or these could be introduced at a suitable time to apply the electricalfield needed to render the conducting or semi-conducting structuresbipolar thus stimulating electrochemistry. This would result in theproduction of a detectable signal. The multiwell plate may also beconstructed using composite materials containing conducting and/orsemiconducting structures of the subject invention. Multiwell plates soconstructed would possess well surfaces of conducting and/orsemiconducting structures able to act as bipolar electrodes in an appliedfield.
It will also be understood that the structures of the invention assuspended structures in assay buffers may be used in a multiwell plateformat in addition to the potential to include them in the plate structure.
In a multianalyte system, the conducting or semi-conductingstructures of the subject invention could take the form of multipleindividual patterns or shapes on the surface of a non-conducting materialforming a multitude of potential assay sites rendered bipolar in an appliedelectrical field. These bipolar structures of the subject invention would then be capable of electrochemistry in electrolytes known to the art. Theresult of this electrochemistry would be to allow production of adetectable signal dependent on the presence of an electrochemically activespecies in proximity to the bipolar structures of the subject invention. Inthis multianalyte cell, the surface with its multiple individual patterns orshapes of conducting or semi-conducting material may form the bottom ofa well in an assay plate with many other such surfaces as found in amicrotitre plate or be part of a flow-through system as a means forintroduction of sample and or reagents. In these and other related cells,the surface with its multiple structures must be in contact with anelectrolyte and must have at least one window to allow the detection ofthe detectable species. This provides for significant advantages over thecurrent art which requires external electronic connections to each element.Further examples of cells are described in the section "Assay formats" andthe section "Examples".
During analysis of the sample, a voltage is applied to the sample.The following section describes some examples of this and the waveformsthe applied voltage may take.
Applied voltage profiles can be those seen in typical electrochemicalstudies and cells with the exception that these are not limited to the lowvoltages seen in normal electrochemical cells (less than 5 volts). Thevoltages which may be applied to the structures of the subject inventionrange from 2 volts/cm to 50,000 volts/cm or the limit of thesolvent/electrolyte used. Advantageously, voltages range from 20 volts/cmto 50,000 volts/cm or the limit of the solvent/electrolyte used. Mostadvantageously, the voltages range from 100 volts/cm to 50,000 volts/cmor the limit of the solvent/electrolyte used. The voltage applied to the cellof the subject invention via the externally and electronically connectedelectrodes can take multiple forms. For example, the voltage may beapplied in a step form where the potential is raised to a given value and held for a time and then removed. This type of step may be appliedrepeatedly to give a square wave form with a wide range of frequenciesfrom 0.001-10,000 cycles per sec (advantageously this is from 0.01 to 1,000cycles per sec). The voltage may also contain portions which apply reversepotentials. Thus the step potential may be described as a negative and thena positive portion and this single unit may be repeated to give analternating square wave form. The voltage may also be applied in such away that it follows a ramp up and down in voltage with or withoutnegative or reverse polarity portions to the ramp. These ramps may beconstructed around numerous potential waveforms such as sin waves, sawtooth waves, multiple component step waves and combinations of these.Also, it may be advantageous to have at least a lower voltage portion insuch wave forms prior to the excitation of the electrochemically detectablespecies. Where a single cycle of applied voltages is used, this cycle maytake from 0.01 to 1600 secs to complete, advantageously this may be from1 to 60 seconds.
The measuring cycle in the instrument consists of the introductionof the sample into the central zone of the cell either by flow (in a flow-throughsystem), by robot arm or other manual or mechanical pipettingmeans, or by placing a cartridge and/or disposable element containing theelectrodes, conducting and/or semi-conducting structures and sample intothe instrument. The sample is then subjected to a voltage waveform orpulse as outlined above and the detectable electrochemical reaction is thendetected.
The sample may be subjected to sonics or ultrasonics to improve thekinetics of the binding reaction and/or the electrochemistry as has beendescribed previously for these reactions and is known in the art (KSuslick, Science, 1990, 247, 1439-1445 and D Walton et al ElectrochimicaActa, 1993, 38, 307-310). Mixing may also be achieved via the use ofmagnetic methods or other mechanical methods known in the art which are known to achieve significant fluid mixing. These various forms ofmixing can be advantageous in that the mixing during the application ofthe applied voltage, activating the electrochemistry, can induce tumblingand rotation of the structures of the subject invention is such a way thatsignificantly more surface of the structures is activated to carry out thedesired detectable electrochemical reaction(s).
The voltages and waveforms described above may also be appliedduring mixing or sonication or ultrasonication. In addition, multiple(advantageously from 2 to 20) externally and electronically connectedelectrodes may be used in a cell to achieve the application of a voltagefield at various angles with or without the application of alternatingpositive and negative fields to achieve activation of a greater surface of thestructures of the subject invention. In one of its forms, externally andelectronically connected electrodes could be positioned at the eightpositions of a sphere, or corners of a cube or rectangle defining the centralzone of the cell. These electrodes could then be used in pairs or othervarious combinations to apply both negative and positive voltages to thecell. These voltages could be controlled by a computer or mechanicaldevice to apply a sequence of voltages to the different electrodes in such away that all faces of the conducting and/or semi-conducting material areactivated to carry out the desired electrochemistry to produce thedetectable signal. Activation of various faces of the conducting and/orsemi conducting structures of the subject invention could also be used incombination with multiple detectors of electromagnetic radiation topermit correlation of applied voltages and electromagnetic radiation fromvarious faces to be made thus allowing a more effective determination andelimination of background signals in the system and from the detectorsand their associated circuits.
The various forms in which the electrochemically-detectable signalcan be detected are numerous and are known in the art, for example: inthe case of ECL and chemiluminescence, a light detection method based on the use of photomultiplier tubes and photodiodes (luminometers), CCDcameras, film and by eye; in the case of a fluorescent molecule generatedby the electrochemistry, a detector based on a fluorimeter would be used;in the case of a chromogenic or other molecule with a new absorptionspectrum, a spectrophotometer system would be used; in the case of anelectrochemically active molecule generated as the electrochemicallydetectable signal, an electrochemical cell with externally electronicallyconnected electrodes would be used to electrochemically detect the speciesgenerated using potentiometric or amperometric methods.
Various assays are described for the subject invention and these canbe carried out making use of numerous binding species. The following areexamples illustrative of the types and kind of binding species contemplatedin the subject invention. In this discussion, a molecular species includeschemicals from single species atoms or ions (i.e. Ca2+) to complex multipleatom compounds (i.e. antibodies). Most binding species are defined by theexistence of a partner binding species since, to be a binding species, themolecular species must bind to another molecular species which may bethe same molecular species. For example, an antibody may be developedsuch that it will bind to itself. In many cases, this type of self binding isnot valuable but can be useful in certain cases, for example, wheredevelopment of signal amplification is of interest. In the case of a bindingspecies and its binding partner, these two species can be considered to bebinding species since they are both able to bind to another molecularspecies. For example, in the case of antibodies and antigens (well knownbinding species), the antibody binding species' partner is the antigen andthe antigen binding species' partner is the antibody. Thus, the antibodyand the antigen can both be considered to be binding species. Thefollowing is a partial list of such binding species contemplated in thesubject invention: antibodies, antigens, receptors, enzymes, enzymeinhibitors, drugs, hormones, ligands, receptor ligands, lectins, selectins, integrins, RNA, DNA, nucleic acid analogues, nucleic acid bindingproteins, chelating ligands. These binding species are all contemplated asanalytes that can be detected and/or quantitated by the subject invention.
Analytes of the subject invention are numerous, including thebinding species described above, but also including molecules which maynot be binding species or can be detected without being part of a binding-speciesto binding-species interaction. The character which defines thesegroups is their ability to interact with the electrochemistry in a detectablemanner, this interaction occurring because of the applied voltage on thestructures of conducting and/or semi-conducting materials not in externalelectronic contact. Also considered as analytes are molecules able directlyor indirectly to produce, destroy or metabolise electrochemicallydetectable species. An example would be the detection and/or quantitationof amine-containing compounds which act as a coreactant in the generationof ECL from various metal chelates such as Ru(bpy)32+. Examples of thisare contained in EP 0 441 894 B1 which is incorporated by reference.Other examples are tripropylamine, NAD(P)H, and hydrolysed betalactams. It will be understood that ECL labels (as described earlier) andECL active species such as Ru(bpy)32+, ruberene, luminol, and dioxetanesare also considered as analytes. No binding interactions occur in thisreaction. Also, in the case of glucose detection and/or quantitation, whereglucose is acted upon by glucose oxidase, the product of this reaction(hydrogen peroxides) act as a coreactant with luminol to electrochemicallygenerate light. It would be understood from such a reaction that inaddition to glucose as an analyte, glucose oxidase and hydrogen peroxidecould also be analytes. It will be understood that an analyte may besubjected to a series of reactions giving rise to a molecular species whichcan directly participate in modifying (enhancing or inhibiting) a detectableelectrochemical reaction. An example of this is seen in the case of aglucose assay based on a well understood assay for glucose which first kinases the glucose (with ATP and the enzyme hexose kinase) to formglucose-6-phosphate. This is then subjected to the action of glucose-6-phosphatedehydrogenase in the presence of NAD+ to generate NADH.This acts as a coreactant with Ru(bpy)32+ to generate ECL (Jameison F, etal. 1996, Anal Chem 68, 1298). Assays based on coupling multiplereactions and/or chemical reactions such as that described for glucose areknown and well understood and those reactants, enzymes and substrateswhich participate to give rise to an electrochemically detectable reactioncan be considered as analytes. Thus, it will be understood that many otherchemicals may be analytes in addition to the binding species describedabove. Some example of analytes include enzyme substrates, enzymeproducts, enzymes, whole cells, viruses, sub cellular particles, nucleicacids, polysaccharides, proteins, glycoproteins, lipoproteins,lipopoylysaccharides, lipids, fatty acids, peptides, cellular metabolites,hormones, pharmacological agents, tranquillisers, steroids, vitamins,amino acids, sugars, and non biological polymers.
Numerous assay formats are contemplated and are given in the formof examples below.
The assay formats of the subject invention provide variousadvantages over the current assay systems. Also provided are new ways ofcarrying out analyses which allow for a series of contemplatedimprovements to assay systems and scientific exploration.
One key advantage over the current electrochemically baseddetection or assay systems - where the electrochemistry is confined to thesurface of an externally electronically connected electrode - is that thesubject invention does not present any such limitation. The subjectinvention allows for electrochemical stimulation within a sample volume,generating a detectable signal which allows for the detection and/orquantitation of analytes within the sample volume and to a greater extentthan previously possible because of the effective extension of the electrode surface area available. This ability to generate a detectable signal bystimulation of electrochemistry within the volume of a sample providesthe following advantages :
- 1. Improved detectability, more of the sample is activated,
- 2. Improved sensitivity, more signal is developed as the sample volumeis significantly larger than in conventional electrochemical methods,
- 3. Improved speed, improved kinetics and time to analysis,
- 4. Increased volumes for samples allows activation through the samplevolume,
- 5. Spatial discrimination, allows multiple samples to be analysed andprovides the potential of photographic-like resolution within asample i.e. a gel. This spatial discrimination can also be threedimensional,
- 6. Improved electrochemistry via redox cycling especially whenstructures of the subject invention are coupled to or linked via abinding interaction which is mediated by an analyte of interest, thusallowing detection of the binding reaction without the need forseparations or washes following the binding interaction.Assays in separation media.
These advantages of the subject invention are significant whendetermining the location of labelled material localised within a largevolume or surface. Examples of this are electrophoretic separations,chromatographic separations (TLC, HPLC, FPLC),immunochromatography, southern blots, western blots, northern blots. Inthe case of electrophoretic separations, where the detection of thepositions and amounts of each species within the gel is of interest, variousmethods are used for detection and quantitation, i.e. staining with variousdyes and fluorescence. Typically, this results in staining the gel directlywith various agents to visualise the molecules in the gel, e.g. usingcommassie blue to detect proteins in gels. Here, the proteins bind the dyeand are detected as blue bands. Ethidium bromide is also used to detect DNA in gels followed by illumination with UV light to visualise thefluorescence of the DNA-bound ethidium bromide. In some cases, themolecules are radiolabeled and are detected by autoradiography. In thecase of this invention, it is possible to manufacture gels which containstructures (i.e. particles, fibres, fibrils etc.) of conducting material whichcan be activated to carry out electrochemistry to allow the detection ofanalytes separated by the gel electrophoresis. Numerous examples of gels,papers and other electrophoretic supports are known in the art e.g.polyacrylamide, agarose, starch, cellulose acetate, cellulose, silica, nylon,nitrocellulose, PVDF, etc.
In an advantageous embodiment of this gel detection application ofthe invention, analysis of PCR products could be carried out as follows.Oligonucleotide primers are labelled with an electrochemiluminescent(ECL) label ORIGEN TAG (IGEN, Gaithersburg MD) following methodsdescribed by the manufacturer (application notes describing PCR areavailable on the internet at http://www.igen.com). These PCR primers arethen used in a PCR to amplify and generate an ECL-labelled PCR productwhich is dependent on the presence of DNA complementary to thelabelled PCR primers. The PCR product can now be run on a standardagarose or polyacrylamide gel containing for example advantageouslycarbon nanotubes (Materials and Electrochemical Research Corp, Tucson,AZ) as conducting and/or semi conducting material. These nanotubeswould be included at such a concentration that the gel is not conductiveand is not totally opaque such that no ECL would be visible,advantageously 0.1-10 µg/ml. It is understood that other suitableconducting or semi-conducting material (as described above) may be usedin the gel. The gel is then loaded with the PCR products and the DNAsubjected to electrophoresis at voltage gradients typical for the separationof PCR products i.e. less than 100v/cm. After the electrophoreticseparation, the gel is then soaked in ORIGEN assay buffer (IGEN,Gaithersburg, MD) for 30-60 min with three changes of buffer. This gel is then placed in a high voltage chamber contained within a black box toexclude as much light as possible with electrolyte in contact at each end ofthe gel as in a typical electrophoresis chamber. The high voltage containerhas an electrode at each end as is typical of an electrophoresis chamberwith the gel between these electrodes. The gel is visualised by aphotomultiplier tube, film or CCD camera. The door to the black boxcontaining the gel, high voltage chamber and photon detection system(PMT(s) or CCD camera or photodiode(s)) is closed to create asubstantially light-free environment. The high voltage chamber is thensubjected to a high voltage which typically increases at a rate of 100 v/s to10 kv/s but could also be in the range of 10v/s to 1,000 kv/s. This voltageramp is applied for 0.1 sec or longer. During the application of these highvoltages, peak voltages are controlled to prevent arcing which wouldindicate an excessive voltage. The voltages reached are advantageouslyfrom 100 v/cm to 10,000 v/cm. During the high voltage sweep the lightproduced is detected by the CCD camera (or other light detection means)and the location and/or amount of the PCR product is determined.
In a further embodiment of the above protocol, the electrophoresisbuffer would also be the ECL buffer as in the case of a buffer (as describedearlier) which contains amines useful for the generation of ECL, forexample tripropylamine-phosphate, tripropylamine-acetate, triethylamine-borate,tripropylamine-borate, PIPES and HEPES (WO 97/33176).Commercial examples of ECL buffers are ProCell (Boehringer Mannheim)and ORIGEN assay buffer (IGEN). Alternative methods for ECL couldalso be used where different pHs may be desired for electrophoresis, forexample by the use of the tris(2,2-bipyridyl) ruthenium oxalate system. Inthis case, where the ECL enabling buffer and/or co-reactants are present,the gel could be pulsed to the higher voltages at desired times during thelower voltages used for the electrophoretic separations to activate the ECLlabels on the labelled molecules. The higher voltages are required torender the carbon nanotubes or fibrils bipolar (generating an anodic and cathodic face on at least some of the carbon nanotubes or fibrils, or otherstructures of conducting and/or semi conducting material) resulting in theactivation of the ECL label contained in the PCR products separated onthe gel, as described above, or other labelled molecules. This high voltagepulse used to render the carbon nanotubes or fibrils bipolar and activatethe electrochemistry on part of the surface of at least some of the carbonnanotubes or fibrils could thus be part of the electrophoresis protocol.This would allow the electrophoretic migration to be followed over timeby intermittent activation of the ECL labels attached to the labelled PCRproducts. Thisin situ monitoring of the electrophoresis would bevaluable, improving the speed and resolution of the electrophoresis. Theseelectrophoretic methods, of great interest for the analysis of biomolecules,could also be applied in the analysis and detection of ECL labels notcoupled to biomolecules. The application of this method to the analysisand detection of ECL labels and ECL coreactants not coupled tobiomolecules could also be extended to ECL active compounds such as Ru(bpy)32+ and to compounds able to stimulate, enhance and support ECL,such as tripropylamine.
A further modification of the above example concerning thein situdetection of an analyte of interest within gels or other separation media isalso contemplated. This method contemplates the detection of numerousmolecules able to support ECL as has been demonstrated for numerousamine-containing molecules such as TPA, TEA, PIPES, HEPES, beta-lactams,NADH, and enzymes coupled to NAD or NADP etc. Thisprovides the potential to detect enzymes and substrates of enzymes eitherdirectly or indirectly via a series of enzyme reaction steps (Martin AF etal, 1997, Biosensors and Bioelectronics 12, 479-489, GA Forbes et alAnalytica Chimica Acta 347, (1997) 289-293). This is also possible withenzymes that give rise to hydrogen peroxide, e.g. glucose oxidase, cholineoxidase etc., the resultant hydrogen peroxide being detected via couplingto the ECL of luminol. This luminol ECL can also be coupled to the detection of hydroperoxides such as lipid hydroperoxides. In this format,the conductive or semi-conductive material may be precoated with an ECLactive label sensitive to the presence of amines or other potentialcoreactants that enhance ECL, e.g. Ru (2,2-bipyridyl)32+ when theseamines or other potential co-reactants are in close proximity to thedispersed conducting or semi-conducting material coated with the ECLactive species. This type of detector system would be useful in thedetection of materials in gels, material eluting from columns duringpurification, during capillary electrophoresis and obviate the problemswith the application of conventional electrochemical stimulation whichrequires sample within a narrow region of an externally electronicallyconnected electrode surface which, being able to analyse or activate only avery small portion of any given sample, results in poor sensitivity forthese conventional methods. Here, the coated conducting or semi-conductingmaterial would be excited by the application of an externalvoltage to generate bipolar faces on at least some of the dispersedconducting or semi-conducting material, and thereby allow the detectionof the ECL co-reactants such as amines, beta-lactams, NADH, NADPHand peroxides. In a further variation of this method, the ECL activespecies may be added in solution i.e. in the gel, HPLC or capillaryelectrophoresis buffers. This would remove the need to immobilise theECL species to the conducting and/or semi-conducting structures of thesubject invention. Alternatively, this system could be used to detect ECLlabels directly or by supporting ECL with a co-reactant. In the case of theluminol peroxide system, peroxides could be detected e.g. hydrogenperoxide or lipid hydroperoxides.
In an alternative embodiment of the above example, theseconducting or semi-conducting materials coated with an ECL active speciessensitive to co-reactants such as amines, peroxides, beta-lactams, NADHand NADPH, could be used as suspended electrode systems for thedetection of various molecules which affect the concentration of these co-reactants. For example, enzymes acting on various substrates can affect theconcentration of these co-reactants allowing detection of either theenzyme or the substrates of the enzyme. An example would bedehydrogenases that generate NADH or NADPH, e.g. glucose-6-phosphatedehydrogenase which can be used in a glucose assay when coupled tohexokinase as described earlier. In this system, the analyte of interest is amolecule which can be specifically acted on by a dehydrogenase to produceNADH or NADPH. This is then detected and quantitated by theactivation of ECL. In the previous example this is glucose-6-phosphate.The assay could be carried out as follows: the sample would be mixed withthe appropriate dehydrogenase and the appropriate co-factor such as NADor NADP. This enzyme/substrate/co-factor mix would then besupplemented with particles, fibres or fibrils of conductive or semi-conductivematerial precoated with an ECL active label sensitive to thepresence of NADH or NADPH, e.g. Ru (2,2-bipyridyl)32+. Alternatively,the conductive or semi-conductive material could be added with an ECLactive species such as Ru (2,2-bipyridyl)32+ free in solution. After theconductive or semi-conductive material and the ECL active speciessensitive to the NADH or NADPH co-reactant have been added, a voltageis applied. This results in the production of bi-polar faces on theconductive or semi-conductive material leading to ECL which is dependenton the concentration of the analyte of interest. In addition to the potentialto immobilise the ECL active species, it is also possible to immobiliseadditionally the enzyme and the co-factor. These systems - usingconventional electrochemical methods - have been described by Martin AFet al, (1997, Biosensors and Bioelectronics 12, 479-489). It is also possibleto add the ECL active species into the solution, not immobilised on theconducting or semi-conducting structures of the subject invention.
These examples of embodiments of the invention concerning thedetection of molecules within a large volume of sample (not possible withcurrent electrochemical detection methods) are directed at ECL but could also make use of other detectable signal systems such as fluorescence,chemiluminescence or spectrometric methods as described in othersections of the invention. These alternative detectable signal systems areactivated by the EC that itself is induced by the application of elevatedvoltages, inducing the appropriate bipolar character on the conducting orsemi-conducting materials within the bulk or volume of the sample.
Assays using filter wicking and/or immunochromatography
In an advantageous embodiment of the subject invention, thedetection of antigens, antibodies, receptors, ligands and nucleic acids andtheir analogues is possible. This embodiment relates to an immunoassay,nucleic acid assay or other similar binding assays carried out using a filterstrip or wicking method and also called immunochromatographic tests orlateral-flow tests. Examples of these are found in US 5591645, US 4861711,EP 0 258 963 A2, US 4703017 and US 5008080 which are herebyincorporated in entirety by reference. This embodiment relates to theelectrochemical activation of at least part of such a filter or flow-supportingmaterial such that an analyte contained within or boundthroughout the body of the filter or flow-supporting material is detectedand or quantitated. These tests using a filter strip or wicking method andalso called immunochromatographic tests or lateral-flow tests, offer thebeauty of simplicity. They involve simply the application of a sample (e.g.blood, urine, saliva). The application of the sample results in the driedlabelled antibody and the other assay reagents dissolving and mixing withthe sample to start the antibody-analyte binding reaction. During thisbinding reaction, the sample and labelled antibody is wicked (via capillaryflow) along a strip which, at a predetermined point, contains immobilisedsecond antibody for the analyte of interest. The result is that as the sampleand labelled antibody is transported along the membrane or supportingphase, the analyte binds the antibody. This immune complex then at somepoint is transported in the liquid flow to the capture antibody which isimmobilised at a given site within the filter or supporting phase. When the analyte-labelled antibody complex interacts with the immobilised captureantibody it is captured by binding to an available epitope on the analyte ofinterest, resulting in the immobilisation of the label which is attached tothe labelled antibody and complexed with the analyte. The fluid continuesto wick or flow past the immobilised antibody carrying any un-complexed(not bound to analyte of interest) labelled antibody away from theimmobilised capture antibody. At this point, typically, the labelledantibody is detected. These assays use a number of different labellingmethods such as particles and enzymes. In application of theseimmunochromatographic methods, as an advantageous embodiment of thisinvention, an ECL label could be bound to the antibody (IGEN, mc,Gaithersburg, MD) although other labels are possible, and the filter matrixor supporting phase could be manufactured to include structures ofconductive and/or semi conductive materials. Examples of such conductiveand/or semi conductive materials are carbon nanotubes, carbon fibres,glassy carbon particles, Au and/or Pt nanowires. These conductivematerials would be mixed with the materials used in the manufacture ofthe filter membranes or fibre wicking material in such a way that theywould be immobilised within the bulk of the membrane or supportingphase in such a way that the supporting phase or membrane is nottransformed into an electronic conducting or semi-conducting material.
An alternative embodiment would be to immobilise the captureantibody onto a conductive or semi-conductive material (i.e. glassycarbon) and to impregnate the filter or supporting material with theseparticles at a given site. An example of this is described in US 5008080,but only uses plastic microparticles as a solid phase for antibodiesimpregnated into a glass fibre filter for use as a capture phase in anenzyme based immunoassay. These antibody-coated particles could, forexample, be ink jet printed onto the filter or supporting material orpipetted. With a suitable selection of particles or fibres, nanotube sizes oraggregates, these would not migrate from the site of impregnation due to the relative size of the antibody-coated particles to the pores or channelsin the filter or flow-supporting material. Alternatively, the conductiveparticles or fibres could be chemically or physically trapped in the filteror flow-supporting material by impregnation of the conductive or semi-conductiveparticles or fibres using printing or injection methods followedby antibody immobilisation either onto or within close proximity of theconductive or semi-conductive material. Potentially appropriateconductive and semi-conductive materials are carbon nanotubes, carbonfibres, C, Au and or Pt nanowires. With this combination of an ECL-labelledantibody and an electrochemically activatable filter or flow-supportingmaterial, the assay would be carried out as described above forthe traditional filter strip or wicking method (also calledimmunochromatographic tests or lateral-flow tests) but the detection ofthe antibody-captured analyte/antibody complex would be achieved viathe application of a high voltage field (advantageously 2v/cm to 50 kv/cm,most advantageously 500v/cm to 50 kv/cm) across the filter or flow-supportingmaterial such as to generate electrochemically active portionsof the surface resulting in the generation of light via ECL. The ECL lightis detected via a number of understood methods for light detection and orquantitation, including eye, photography, photodiode, photomultiplier orphotocell.
It will be understood by those skilled in the art that this method ofanalyte detection using a binding reaction between antibody and analytecould also be applied without a labelled antibody but via the use of alabelled analyte which competes with the sample analyte for binding to thecapture antibody. It is also understood by those skilled in the art thatthese immunoassay formats are applicable to numerous binding assaysbased on other well understood binding interactions, e.g. nucleic acid -nucleic acid, receptor-ligand, nucleic acid analogues-nucleic acid, andnucleic acid-protein. It is also understood that these immunochromatographic assays may be developed with liquid reagents notdried onto the immunochromatographic assay device.
In a further embodiment of the above method where the captureantibody or nucleic acid is attached to the conducting or semi-conductingmaterial dispersed throughout at least a portion of the flow or filtermaterial; the assay may be carried out without the need for a so-calledwash step to remove the unbound or free label. In this case, the binding ofthe labelled species modulated in the presence of the analyte is detecteddue to the effective concentration of the label on the surface of theconducting or semi-conducting material which allows selective ECactivation of the bound labelled species over that of the unbound. Thismethod - which allows the assays to be carried out without a wash - is ofgreat benefit in that it allows for rapid testing and simplification of thediagnostic system device.
In a further embodiment related to using a filter strip or wickingmethod (also called immunochromatographic tests or lateral-flow tests),the structures of conductive or semi-conductive material - advantageouslywith the longest dimension being less then 20µm - would be the label and abinding species attached to this label. In this embodiment, the assay wouldbe conducted in the normal manner for such immunochromatographictests or lateral-flow tests, resulting in the capture of the structures ofconductive or semi-conductive material on the capture antibody at apredetermined site in the immunochromatographic tests or lateral-flowtests strip or cartridge based on the presence of analyte. Such assaysalready exist and make use of latex beads, colloidal gold or silver as a labelon the antibody (Boehringer Mannheim) but these use visual detection ofthe particles and no use is made of the electrochemical properties of theconducting or semi-conducting material.
In this invention, we would carry out the immunoassay in a similarformat but make use of, for example, a buffer containing ECL activespecies. When the binding reaction and capture to the immobilised binding species (antibody) is completed, the labels - which are conductive or semi-conductivematerials - would be activated. The activation of the boundconductive or semi-conductive material would be achieved by theapplication of a high voltage (advantageously 2v/cm to 50 kv/cm, mostadvantageously 500v/cm to 30 kv/cm) across the binding zone in theimmunochromatographic tests or lateral-flow tests. This application of ahigh voltage across the binding zone results in the formation of cathodicand anodic faces on the labels of conductive or semi-conductive materialsused in the immunochromatographic tests or lateral-flow tests, andgenerates bipolar labels. The result of the formation of these bipolar labelsis that they become electrochemically active and result in the activation ofthe ECL active species and the emission of light. This can be detected innumerous ways as described in this invention. It is also possible togenerate other detectable signals using this format, e.g. fluorescence,chemiluminescence using luminol or acridinium esters which is a wellunderstood method for the generation of light from an electrochemicalreaction. Stabilised dioxetanes are also considered since they can beactivated by electrochemical methods. Visible signals can also be generatede.g. by electropolymerisation to generate polypyrole and polythiophenes.The conductive or semi-conductive material used as the label may also beelectroluminescent or chemiluminescent without addition of active speciesto the buffer used for the assay. An example of such an electroluminescentor chemiluminescent material is Al (Haapakka K et al, 1988, Anal ChimActa 207, 195-210). It is also contemplated that other electroluminescentcompounds may be used in this way. This mode of detection for theconducting or semi-conducting label is superior to known visual methodsas the labels can be detected before they are visible or they can beamplified to become visible as in the case of electropolymerisation. Thus,the invention improves on the current immunochromatographic tests orlateral-flow tests which use conductive or semi-conductive materials aslabels.
In an alternative embodiment, these filter wicking assays may alsobe applied to the detection of analytes without the need for a bindingspecies. In these assays, ECL labels or co-reactants may be detected.Examples of potentially-detectable analytes have already be describedabove for the subject invention. A good example would be the detection ofglucose, where the application of the sample to the filter-wicking substratebrings the glucose into contact with glucose oxidase and luminol. Theglucose in the sample is acted upon by the glucose oxidase to generatehydrogen peroxide and this wicks up the wicking substrate and comes intocontact with the structures of conducting and/or semi-conducting materialof the subject invention. The wicking substrate with the sample is thensubject to an applied external voltage which activates the structures ofconducting and/or semi-conducting material to generate ECL from theluminol and the hydrogen peroxide produced by the action of glucoseoxidase on the glucose in the sample. This ECL is used to detect and/orquantitate the glucose in the sample. The value of a glucose assay in thisformat is that it allows the separation of the sample prior to the enzymereaction and detection of the ECL. This is most valuable as, for example inthe case of a blood sample, it allows the removal of the red and whiteblood cells and other interfering substances. This glucose assay may alsobe carried out with dehydrogenases using the ECL from the Ru(bpy)32+and NAD(P)H reaction using the coupled enzyme reaction employed forglucose analysis of hexokinase and glucose-6-phosphate dehydrogenase (asdescribed above in analytes).
These filter wicking assays are thus not restricted to the detection ofbinding reactions but can be used to detect enzymes, substrates ofenzymes, ECL co-reactants, ECL labels, ECL active molecules and ECreactive species as described earlier for the subject invention.
Assays making use of 1-10µm carbon structures (particles) in freesolution.
An advantageous embodiment of the present invention is a bindingassay such as an immunoassay, receptor, ligand or nucleic acid assay wheresmall conducting or semi-conducting structures are coated with one of thebinding species to form the capture phase of the assay. This coating maybe made in numerous formats known in the art. Advantageously, glassycarbon (or graphitic) structures 1-50µm in diameter are activated bystandard methods to generate, for example, NHS ester-activated structures(L. Dong et al 1996 J Mol, Rec. 9, 383-388). These NHS ester-activatedstructures are then allowed to react with the binding species of interestsuch as an antibody, receptor, ligand, antigen or nucleic acid. This resultsin the generation of a binding species coated onto the glassy carbonstructure. Typically, any non specific binding in the assay is blocked byincubation with a non specific protein such as BSA and/or an amino-containingcompound such as ethanolamine which react with anyremaining NHS groups and bind to non-specific binding sites on theparticle surface. These glassy carbon particles coated with a specificbinding species such as an antibody (or streptavidin) and blocked are nowready to be used as the solid phase in a binding assay. Typically, theseparticles are added to a mixture of sample and another binding specieswhich is labelled with an EC activatable label and able to bind to theanalyte of interest. This mixture is allowed to incubate typically for 1-60min. During this time, the labelled binding species, for example anantibody, binds to the analyte of interest and this complex is thencaptured by the binding species immobilised on the surface of the glassycarbon particles. This series of binding reactions results in theimmobilisation of the EC activatable label on the surface of the glassycarbon particle which is then available to be activated. In one method forthe activation, detection and/or quantitation of the bound label, thesample is now directly activated by the application of a voltage such thatbipolar faces are generated on at least part of at least some of the glassycarbon particles. The bipolar faces so generated are of sufficient voltage that they are able to activate the EC activatable label(s). Typically, the ECactivatable label is an ECL label which is detected using standard methodsfor the detection of light, e.g. PMT, photodiode, film, eye, CCD etc. In analternative method of the above assay, the glassy carbon particles are firstwashed with a suitable buffer prior to the activation of the EC activatablelabels as described above. The washing of the glassy carbon particles couldbe achieved in various ways. Using magnetic beads as the basis for theassay would allow for a method based on magnetic capture, butcentrifugation and filtration are potentially valuable methods which couldbe applied. In the case of filtration, the beads could be readily collectedand washed on the surface of a filter which could form the matrix onwhich the beads could be activated by the application of a voltage acrossthe filter. The application of a voltage across a filter could make use ofany axis and said axis of applied voltage may also advantageously bechanged, rotated or scanned and the polarity of the field changed such thatmore of the captured structures and/or more of the structures coated withimmobilised EC activatable labels are activated. For example, analternating voltage may be applied with various frequencies and waveforms(or pulses). Additionally, the application of sonics may be usedadvantageously to improve signal generation, binding kinetics (i.e. antigenand antibody) and electrochemistry as has been demonstrated previously(Malins et al 1997, J Phys Chem, 101, 5063-8). These sonics may be from 1to 100,000 Hz (cycles per sec), covering the range from sub-sonics toultrasonics. Additionally, sonics can improve signals by inducing tumblingof the structures. This results in more of the surface being subjected toelectrochemical activation. This assay format is ideal for all sandwichimmunoassays and DNA probe sandwich assay formats. Also, thestructures of conducting and/or semi conducting material may have aparamagnetic component allowing capture for washing and/or optimalpositioning of the structures with respect to the voltage field and/ordetector.
An alternative embodiment of the assay described above for glassycarbon beads is an assay that detects an analyte which is in competitionwith the bead-bound binding species for binding with a labelled equivalentof the analyte. The label in this example is the same as described above,being an EC activatable label which results in the production of adetectable signal or EC detectable species. In such an assay, the sampletypically will have added to it the labelled equivalent of the analytefollowed by the addition of the beads or other conducting or semi-conductingmaterial coated with a binding species specific for the analyteof interest or capable of binding to a binding species specific for theanalyte of interest. This mixture is then allowed to react typically for 1-90min during which the analyte and the labelled analyte equivalent competefor binding to the immobilised binding species. During this competitionstep, the amount of bound labelled analyte equivalent is inverselyproportional to the level of the analyte in the sample. Following thisbinding step, the bound electrochemically activatable and/or detectablelabel or moiety is detected as outlined in either of the two methods givenabove.
An example of such an assay for oestradiol is outlined below in theexamples.
Scanning or alteration of the polarity and position of the bipolarfaces may also be used to specifically generate ECL by rapidly generatingoxidised and reduced species (i.e. Ru3+ and Ru+). These are then able toreact to yield the excited state which can then emit electromagneticradiation. Also, the frequency of the applied voltage may be varied from 0to 200,000 cycles per second, and advantageously from 0 to 5,000 cyclesper second, to enhance certain reaction mechanisms, e.g. in the doublepotential step mode of ECL (Edge et al), and in luminol hydrogenperoxide electrochemically activated chemiluminescence.
It will be understood by those skilled in the art that numerouscombinations of conducting and semi-conducting structures are possible and numerous assays are possible ranging from immunoassays, receptorligand assays and nucleic acid probe assays to those used to detectsubstrates for NAD- and NADP-dependent enzymes, oxidases aminesperoxides, oxalates and other ECL active molecules or co-reactants. In thecase of the binding assay, the various formats give rise to the followingcompositions in the presence of analyte during the specific binding step(s);
- 1) structure-SA-Bio-Ab-At-Ab-El + Ab-El
- 2) structure-Ab-At-Ab-El + Ab-El
- 3) structure-At-Ab-El + At(in sample)-Ab-El
- 4) structure-Ab-At-El + structure-Ab-At + At + At-El
- 5) structure-SA-Bio-At-Ab-El + Ab-El + At(in sample)-Ab-El
- 6) structure-SA-Bio-Ab-At-El + structure-SA-Bio-Ab-At
- Ab;
- is selected from at least one of the following; antibody, nucleic acid,molecular species capable of binding in a sequence-dependent fashion to anucleic acid sequence a so-called nucleic acid analogue or other nucleicanalogue, or a receptor or ligand.
- SA;
- is selected from at least one of the following; streptavidin, avidin ora molecular species able to bind biotin or bind to Bio.
- At;
- is selected from at least one of the following; the analyte of interestwhich may be a ligand or nucleic acid.
- El;
- is the electrochemically active label.
- Bio;
- is biotin, amino biotin, imminobiotin peptide or a molecular speciesable to bind to SA.
- Structure;
- is selected from examples of structures of the subject inventiondescribed earlier.
An example of such assays is described below in the examples forTSH, HCG, oestradiol and nucleic acid assays.
Spatial arrays of multiple zones or elements are also contemplatedby this invention. This consists of conductive and/or semiconductivestructures of the subject invention spatially arranged such as on thesurface of a non conductive surface such as glass or plastics. The methodsfor putting down or coating multiple elements or zones of conductingand/or semi conducting materials are well known in the art, e.g.sputtering, evaporation, electron beam evaporation, screen printing,painting, ink jet printing, other printing methods, photocopying,stamping, physically placing elements and fixing them in place, etc. It isalso well known in the art that methods for coating, etching and recoatingwith conductive and/or semi conductive and non conductive materials areable to produce complex structures with exposed and masked regionswhich contain elements that are also connected electronically via aconductive and/or semi conductive material. It is also contemplated thatspatial multi arrays of conducting and/or semi conducting materials of thesubject invention could also be arranged in a 3 dimensional array, e.g.fabricated into a macroporous support or woven into a 3 dimensionalarray of supporting fibres.
The interest in generating such spatial arrays of structuresconducting and/or semi conducting material of the subject invention is toallow for the construction of an analyte-detection device for multipleanalytes where each of the structures of conducting and/or semiconducting material is a potential electrode capable of carrying out anassay for a predetermined analyte. In this way, the spatial arrays are ableto carry out multiple analyte assays without the need for individualreaction chambers or tubes, or the need for multiple electronic conducting contacts to the structures of conducting and/or semi conducting material.In the array of structures of conducting and/or semi conducting materialvarious materials can be mixed (see above) i.e. Pt, Au, Ag, Cu, C (and itsvarious forms). Also, various methods for making the contemplatedstructures can be used in combination to achieve the optimal combinationsof structures. The use of combinations of coating methods can also be usedto develop combination materials such as platinized carbon by vacuum-depositionmethods for Pt onto screen-printed Carbon structures. Inaddition, various sizes of structures of conducting and/or semi conductingmaterial may be used to allow the sequential activation of various elementsto provide an addition means for determining which assay is being read inaddition to the spatial information from the structures' position withinthe spatial array. This use of various sizes of structures of conductingand/or semi conducting material - which will develop the potentialrequired to activate the detectable electrochemistry of the subjectinvention - is achieved through the dependence of the structure size andthe applied voltage in determining the voltage developed across a givenstructure within the applied voltage gradient. For example, a multi arraydevice might be constructed with strips of carbon paste, (glassy carbonparticles for example from 1-100µm in size, in a plastic or resin binder) orconductive composite. Alternatively, the methods used by Iwasaki may beused to generate thin patterned films of graphitic and conductive carbon(Y Iwasaki, Current Separations (1995) 14, 2-8 and O Niwa,Electroanalysis (1995) 7, 606-613). These strips of composite carbon couldbe various lengths such as 0.1, 0.2, 0.4, 0.8, 1.6 mm. These strips thenplaced into an instrument of the subject invention with the various lengthsaligned such that the various long dimensions of the strips (0.1, 0.2, 0.4,0.8, 1.6 mm) are lined up along an axis advantageously defined by theshortest distance between the externally and electronically connectedelectrodes. This array of potential bipolar electrodes when subjected to anincreasing applied voltage in a solution of electrolyte within the central zone of an instrument of the subject invention would sequentially becomeelectrochemically active. As the externally applied voltage is raised, thelongest conducting structure or carbon composite will become active as itreaches the required voltage across the structures. This will be followed bythe next length, and so on. When using a series of potential bipolarelectrodes 0.1, 0.2, 0.4, 0.8, 1.6 mm long to induce an electrochemicalreaction involving ECL with Ru (2, 2' bypyridyl)32+, the voltage appliedby the externally electronically connected electrodes to the central zone ofthe instrument will double as each electrode becomes activated. In bindingreactions, these various structures of conducting and/or semi-conductingmaterials of the subject invention would be coupled to binding specieswith the potential to present a different binding species on each element,thus enabling multiple analyte detection.
The detection and/or quantitation of multiple analytes on a singlesurface or with a single container is an advantageous embodiment of thesubject invention. In these multiple assay applications, multiple zones orelements are fabricated onto a surface following conventional methodsdescribed for externally electronically connected interdigitated arraymicroelectrodes (Y Iwasaki, Current Separations (1995) 14, 2-8 and ONiwa, Electroanalysis (1995) 7, 606-613) or constructed within a threedimensional structure such as a filter or fabric. Thus, it is envisaged that asample would be introduced into a container, and within this containermultiple zones or elements containing structures of conducting and/orsemi conducting material of the subject invention not electronicallyconnected to an external electronic circuit would allow multiple assays tobe carried out. These assays would be based on the electrochemistryproduced by the structures of conducting and/or semi conducting materialof the subject invention not electronically connected to an externalelectronic circuit when subjected to a voltage gradient in a suitableelectrolyte. Further applications of this embodiment are described below.
The multiple zones or elements for these multiple assays may consistof either a single structure of conducting and/or semi conducting materialforming the zone, or multiple structures of conducting and/or semiconducting material grouped to form the zone or element in a definedregion or portion of an assay component (surface or three dimensionalarray). In the case of the single structures, an example can be given wheregold, carbon or other conducting or semi-conducting material isevaporated, sputtered, deposited, printed, painted or formed onto a surfacemaking a defined shape based on a mask or printing device used in theprocess or by the use of resist patterning and selective etching. Thesedefined shapes are electronically conductive and/or semi-conductivethroughout the defined shape and form the zone or element in which anassay for an analyte may be conducted. In the alternative format, themultiple structures of conducting and/or semi-conducting material areused to from a zone or element for performing a given assay. Examples ofthis are the same as for the previous example but here, the mask orprinting device, or the use of resist patterning and the selective etchingused in the process, allows multiple structures to be deposited or formedin a given zone or element. Alternatively, the multiple structures ofconducting and/or semi-conducting material may already be made and aresimply applied to the surface such as with carbon fibres, fibrils, nanotubesor particles. These various methods result in the formation of multiplezones or elements (each able to carry out an assay) and each zone orelement contains multiple structures of conducting and/or semi-conductingmaterial.
In the case where multiple structures of conducting and/or semi-conductingmaterials are already formed and are then applied to createzones or elements containing multiple structures, these could be appliedusing various printing methods known in the art with or without bindingmaterial to bond the structures to the surface by physical immobilisationwithin a matrix (such as described in example 15 for the filter wicking assays). In the case where no binding material is used to physically trapthe structures of conducting and/or semi-conducting material, these maybe coupled to the surface either covalently or non-covalently. Usingmultiple structures of conducting and/or semi-conducting material withina zone or element to carry out an assay creates multiple bipolar electrodeelements when subjected to an applied voltage field in the presence ofelectrolyte and provides for improved electrochemistry. In the case ofphysical immobilisation within a matrix, such as a filter, multiple analytesmay be analysed in a flow-through or wicking format.
It is also contemplated that multi arrays may also be formed in athree dimensional array, e.g. using arrays of fibres incorporatingstructures of conducting and/or semi-conducting materials of the subjectinvention. In its simplest form, the structures of conducting and/or semi-conductingmaterial could be woven into a fabric to form a filter orwicking material. The incorporation of structures into a fabric or filter isanother example of physical immobilisation. These fibres may be made offibre optical material allowing the lumination and/or collection ofelectromagnetic radiation from these conductive materials within a sampleor within a matrix of such fibres. In this example, the structures ofconducting and/or semi-conducting material may be advantageously coatedonto the optical fibres.
For example, a surface may be patterned with gold and/or otherconducting and/or semi-conducting materials as described above usingvarious methods which are known, to form an array of single structureseach of which acts as a potential bipolar electrode element for a givenassay. In this case the gold forms a single structure of the subjectinvention which is also a single zone or region able to be becomeelectrochemically activated by the application of a voltage field in thepresence of a suitable electrolyte. Also, the surface may have zones which,rather than having a single structure of conducting material forming the zones or elements, may be composed of multiple structures within adefined zone or element for the means of conducting a specific assay.
By way of an illustration; in a practical example, a zone or elementfor carrying out a given assay might be defined as having a size of 5 mmby 5 mm; on a given surface these zones or elements may be spaced 2 mmapart. Thus, when constructing an assay device with 6 such zones, thesurface would be at least 12 mm by 19 mm with the zones or elements in a2 by 3 array. In this form, the 5 mm by 5 mm zones could be made ofevaporated gold, etched carbon, glassy carbon particles or printed carbonnanotubes. The 5 mm by 5 mm zones may be a continuous electronicallyconducting structure but could also be composed of multiple structures of5 µm by 5 µm contained within the 5 mm by 5 mm zones with spacing of 5µm. This would create an array within the 5 mm by 5 mm zones ofstructures of conducting material. A 5 mm by 5 mm zone could contain250,000 5 µm by 5 µm structures of conducting material spaced at 5 µmintervals.
The following example is to further illustrate the detection and/orquantitation of multiple analytes. In the case of glassy carbon particles,there is no need to define the structures of the conducting material on thesurface as the particles already define the structures of the conductingmaterial.Glassy carbon particles 1% (w/v) in polystyrene (10% w/v)dissolved in chloroform are applied to a polystyrene surface (15 mm by 20mm by 2 mm thick) to generate the six 5 mm by 5 mm zones as describedabove. This surface, containing the 5 mm by 5 mm zones, is then etchedusing various methods to better expose the carbon and activate it forcoupling to antibodies using various methods known in the art such asabrasion followed by chemical activation (see examples) or a plasma suchas oxygen (O Niwa, Electroanalysis (1995) 7, 606-613). Antibodies arecoupled to the oxidised carbon surface as described in the followingexamples. Specific antibodies are coupled to the 6 zones by spotting thespecific antibody onto each zone. Following coupling of the antibodies to the zones, the polystyrene surface is blocked with a solution of bovineserum albumin and washed ready for use in an assay. The surface is thencovered with the sample of interest and incubated for 1 hr followed bywashing. The surface is then incubated with antibodies labelled withRu(bpy)32+ for 1 hr followed by washing in PBS. The surface is thenplaced in a cell containing an electrolyte able to support ECL as describedin the examples, and is subjected to a voltage field. The light emitted fromeach of the zones is detected using a CCD camera. The applied voltagefield is advantageously applied in a thin film cell such that the surface ofthe polystyrene coated with the glassy carbon particles is parallel to thevoltage field. The light generated at each of the zones is proportional tothe concentration of the analytes of interest in the sample. In this way,multiple analytes are detected and may be quantitated.
In an alternative embodiment, an assay for nucleic acids iscontemplated. Here, a specific binding species for the nucleic acid is used,typically a nucleic acid sequence but this also could be a nucleic acidanalogue.
Numerous examples of binding assay formats are known for variousnucleic acid-based assays and all are amenable to the assay system of thesubject invention. Some are outlined above in the schematic for bindingassays. In its simplest form, the nucleic acid or DNA of interest (thesample containing the analyte of interest) can be coupled to the surface ofthe structures of conducting and/or semi-conducting material usingmethods known in the art via phosphate, amine, aldehyde or thiol groupson the nucleic acid. The DNA modified structures would then form thebasis of the specific binding surface for the complementary binding nucleicacid or nucleic acid analogue labelled with the electrochemically detectablespecies, for example Ru(bpy)32+. The DNA modified structures are thensubjected to a so-called pre-hybridisation reaction which effectively blocksthe non-specific binding sites on the structure of conducting and/or semi-conducting material. Following this pre-hybridisation, the DNA modifiedstructures are then subjected to the specific hybridisation reaction withcomplementary binding nucleic acid or nucleic acid analogue labelled withthe electrochemically detectable species, for example Ru(bpy)32+. Thishybridisation reaction between the structure-bound DNA and thecomplementary binding nucleic acid or nucleic acid analogue labelled withthe electrochemically detectable species, for example Ru(bpy)32+, can lastfor 1 min to 24 hrs and depends on the concentration of thecomplementary binding nucleic acid or nucleic acid analogue labelled withthe electrochemically detectable species, for example Ru(bpy)32+, thetemperature and the ionic strength of the hybridisation buffer known inthe art. After the hybridisation step, the structures are washed into anelectrolyte able to support the detectable electrochemistry of theelectrochemically detectable species. The structures are then introducedinto the cell of an instrument of the subject invention and subjected to thevoltage field applied to the central zone of the cell by the externallyelectronically connected electrodes. The detectable electrochemicalreaction is then detected, for example by a PMT, CCD camera orphotodiode in the case of a detectable electrochemical reaction based onECL or chemiluminescence. The detection and/or quantitation of thedetectable electrochemical reaction is used to determine the presence andor quantity of a nucleic acid sequence of interest in the analyte or samplenucleic acid. In the case of Ru(bpy)32+ and DNA, the ECL light level isused to determine the presence of a given DNA sample and or the numberof genes present in the sample. It will be understood that thecomplementary binding nucleic acid or nucleic acid analogue labelled withthe electrochemically detectable species, for example Ru(bpy)32+, mayinstead be labelled with a binding species such as biotin and then rendereddetectable by binding of a binding species such as streptavidin coupled toan electrochemically detectable species, for example Ru(bpy)32+.
In an alternative format, known as sandwich hybridisation, aspecific nucleic acid sequence (or nucleic acid analogue) for the analytenucleic acid of interest is coupled to the surface of the structures ofconducting and/or semi-conducting material using methods known in theart via phosphate, amine, aldehyde or thiol groups on the nucleic acid.The nucleic acid modified structures would then form the basis of thespecific binding surface for the analyte nucleic acid of interest. Thenucleic acid modified structures are then subjected to a so-called pre-hybridisationreaction which effectively blocks the non-specific bindingsites on the structure of conducting and/or semi-conducting material.Following this step, the sample nucleic acid containing the analyte nucleicacid is added to these pre-hybridised structures either with, before or afterthe addition of a nucleic acid or nucleic acid analogue labelled with theelectrochemically detectable species, for example Ru(bpy)32+, which issimilarly complementary to the analyte of interest, and subjected tohybridisation.
The nucleic acid coupled to the structure and the nucleic acidlabelled with the electrochemically detectable species are selected such thatthey hybridise to the analyte nucleic acid in such a way that the analytenucleic acid in the sample links the structure-bound nucleic acid to thenucleic acid labelled with the electrochemically detectable species. Forexample, the nucleic acid coupled to the structure and the nucleic acidlabelled with the electrochemically detectable species might be selected tobind to the same strand of the analyte nucleic acid without interferingwith binding of each other to the analyte strand, typically within 1kilobase of each other, and advantageously within 200 bases, mostadvantageously within 50 bases.
This hybridisation reaction of the structure-bound nucleic acid ornucleic acid analogue and the nucleic acid or nucleic acid analogue labelledwith the electrochemically detectable species, for example Ru(bpy)32+, tothe analyte nucleic acid in the sample can last for 1 min to 24 hrs and depends on the concentration of the nucleic acid or nucleic acid analoguelabelled with the electrochemically detectable species, for exampleRu(bpy)32+, the temperature and the ionic strength of the hybridisationbuffer. After the hybridisation step, the structures are washed into anelectrolyte able to support the detectable electrochemistry of theelectrochemically detectable species. The structures are then subjected to avoltage field applied to the central zone of the cell by the externallyelectronically connected electrodes. The detectable electrochemicalreaction, in the case of a detectable electrochemical reaction based on ECLor chemiluminescence, is then detected for example by a PMT, CCDcamera or photodiode. The detection and/or quantitation of the detectableelectrochemical reaction is used to determine the presence and or quantityof a nucleic acid sequence of interest in the analyte or sample nucleic acid.In the case of Ru(bpy)32+ and DNA, the ECL light level is used todetermine the presence of a given DNA sample and/or the number ofgenes present in the sample.
In a further embodiment of the above example, the structures ofconducting and or semi-conducting material are coated, coupled orderivatized with a binding species which binds to a binding species on anucleic acid or nucleic acid analogue. For example, streptavidin could becoupled to the structure and the nucleic acid coupled to biotin. These tworeagents replace the structures coated or coupled to a specific capturenucleic acid in the previous example and allow the hybridisation reactionto occur in solution concurrent with or followed by the capture of thehybrid complex of the three nucleic acids (the binding species-labelledanalyte-specific nucleic acid, the analyte nucleic acid and the nucleic acidlabelled with the electrochemically detectable species). This method alsoallows for the use of a universal structure and a specific captureoligonucleotide which provides for greater flexibility in the developmentof a random access analyser to assay a number of different analytes. This principle forms the basis of the Boehringer Mannheim Elecsys systemwhere the structures (paramagnetic beads) are coated with streptavidin.
Other formats of nucleic acid detection which differ from thetraditional binding reaction-based tests are those where the samplecontaining the analyte nucleic acid is subjected to an enzymatic step whichallows amplification of the amount of the analyte nucleic acid in thesample and/or labelling of the analyte nucleic acid with either a bindingspecies or an electrochemically detectable species. The following are someexamples known in the art.
In the case of the polymerase chain reaction (PCR), one or moreoligonucleotide sequences (primers) are used to prime the synthesis ofnucleic acid resulting in the amplification of the analyte nucleic acidwhich is adjacent to the primers used in the PCR. The product of such aPCR is nucleic acid which can be detected and/or quantitated by the abovemethods. Alternatively, the primers used to amplify the analyte nucleicacid could be labelled with either a binding species or an electrochemicallydetectable species. In the case where the PCR includes a primer labelledwith an electrochemically detectable species, the products of this reactionmay be detected and/or quantitated by hybridisation using structurescoupled to a nucleic acid sequence specific (and complementary) to theanalyte nucleic acid sequence which is amplified by the primer labelledwith the electrochemically detectable species. These structures coupled toa nucleic acid sequence are prepared as described above for the sandwichhybridisation. Also, it will be understood that these nucleic acid-coupledstructures may be substituted with a combination of a structure coupled toa binding species (streptavidin) and a nucleic acid sequence specific (andcomplimentary) to the analyte nucleic acid sequence labelled with acomplementary binding species (biotin) to the binding species coupled tothe structure, as described above. It will be understood that the formatjust described could be modified such that the binding species isincorporated into the PCR and the electrochemically detectable species can be attached to the nucleic acid sequence specific (and complementary)to the analyte nucleic acid sequence which is amplified. It will also beunderstood that in addition to the PCR amplification described, theenzymatic synthesis of nucleic acid using primers may also be used to labelnucleic acid analytes of interest with binding species and/orelectrochemically detectable species using the primer. In addition, it isalso well known that the labelling of nucleic acid with various species ispossible via the use of direct incorporation of labelled NTPs and chemicalmodification of the nucleic acid (US5512433).
In an alternative format, the sample nucleic acid is hybridised to anucleic acid or nucleic acid analogue which is able to form an antigensignificantly different from either nucleic acid in the hybridised form suchthat an antibody will bind the hybrid and allow assay of the analytenucleic acid. In this format, the analyte may be labelled with variousspecies and/or the nucleic acid and/or the antibody specific for the analytenucleic acid to probe the nucleic acid hybrid which is formed. An exampleof such a system is based on the formation of a DNA:RNA hybrid whichis recognised by an antibody specific for the hybrid such as thosedescribed in US 4833084. It will be understood that various combinationsof binding species and labels are possible. It will also be understood thatby analogy, the use of triple helix binding may also achieve a substantiallyequivalent result by recognition of a specific hybrid.
In another nucleic acid assay format, the structures may be coupledto a nucleic acid sequence which is able to hybridise to an analyte nucleicacid either directly from a sample or after amplification using for examplePCR, NASBA or other target-amplification systems. In addition to thesenucleic acid sequences, a probe sequence labelled with an electrochemicallydetectable species complementary to the structure-coupled nucleic acidmay be used. The analyte nucleic acid competes with the binding of theprobe sequence labelled with an electrochemically detectable species forthe structure-bound nucleic acid. A loss of structure-bound electrochemically detectable species results from hybridisation to theanalyte nucleic acid. This results in a signal decrease. An obviousmodification to this method would be to use a binding species on thestructure (streptavidin) and a binding species on the capture nucleic acid(biotin) such that the nucleic acid is captured onto the structure at thedesired time. The hybridisation steps, wash steps and analysis are asdescribed in the examples above.
It is understood by those skilled in the art that the types of samplecontaining analyte nucleic acid of interest include; cells, biological fluidssuch as serum, plasma, urine, saliva, stool, semen, sputum, cell lysates,viruses, bacteria, plant tissue, animal tissues, fungi, achea bacteria, DNA,cDNA, RNA, mRNA, tRNA, rRNA, plasmids, phage, mitochondrialnucleic acid.
Examples of the above formats for nucleic acid assays based onconventional electrochemical activation using externally electronicallyconnected electrodes have been described (Kenten et al 1991 Clin Chem37, 1626; Kenten et al 1992 Clin Chem 38, 873; and Van Gemen et al 1994J. Virol. Methods 49, 157).
In an alternative embodiment of the subject invention, a flow-throughassay system is contemplated. This method is related to methodsused for cell counting and fluorescent cell analysis but provides a novelsolution to improving assay sensitivity and simplicity. In this assaysystem, an instrument is constructed which contains the basic elementsdescribed above containing a voltage control circuit designed to apply avoltage using the externally connectable electrodes which are contained ina cell, a detector for electromagnetic radiation which is a photomultipliertube (PMT), a computer for the control of the voltage circuit, a detectorand a pump. In addition to the elements described previously, theinstrument contains a pump which controls - with the aid of the computer- the flow of the sample, and a cell which is designed to have a narrow zone (the central zone of the subject invention) through which will flowthe structures of the subject invention which are advantageouslyconductive particles. The cell of this instrument in its basic form could bea simple tube with a neck or narrow portion such that the flow of samplecontaining the conductive particles results in the separation of theparticles allowing individual particles to be present in this neck or narrowzone. This basic cell would have externally connected electrodespositioned either side of the central zone composed of the neck or narrowportion of the flow cell. This would place these electrodes up stream anddown stream of the central measuring zone around the neck or narrowportion of the flow cell. Narrowing the flow path in this way creates aflow of single particles past the PMT which is positioned to view thenarrow or neck portion of the flow cell. This narrowing of the flow cell toform a neck also creates an increase in the voltage gradient at this pointsuch that the voltage across the conductive particles reaches a point atwhich a detectable electrochemical reaction can take place. Typically, thisreaction generates light. The other advantage to a cell configured to form agradual narrowing of the flow path is that the voltage gradient willincrease across the conductive particles as they flow through this narrowneck-like region. This gradual increase in the voltage would allow the cellto activate particles of different sizes at different points as they flowthrough the narrowed portion of the cell. For example, the larger particlesrequire a lower voltage gradient than do the small particles to achieve therequired surface voltage to activate any given electrochemical process.Thus, as the larger particles travel in the flow of electrolyte through thenarrowed portion of the cell they will be activated earlier than the smallerparticles to carry out a given electrochemical reaction. This differentialactivation of particles within the flow cell under a given voltage appliedfrom the externally connected electrodes also allows this instrumentconfiguration to detect multiple analytes by determination of the positionat which the particles become activated. For example, an assay would be constructed with particles of various sizes such that each binding speciesfor each assay would be bound to a different sized particle. Each group ofdifferent sized particles would be activated at different positions in theflow cell as the sample flowed through under the voltage applied from theexternally connected electrodes. This mixture of various sized particlescoated with a number of different binding species would be mixed with asample of interest and with labelled binding species to effect a binding ofthe labelled binding species proportional to the concentration of theanalytes of interest in the sample of interest.
For example, each size of conducting particle would be coated with adifferent antibody. This would result in a mixture of particles of varioussizes where a given particle size would have a given antibody coated on it.This mixture of coated conductive particles would then be added to asample which contains a series of analytes to which the antibodies coatedon the conductive particles could bind. In addition to the beads, labelledantibodies or labelled antigen (labelled with an electrochemiluminescentor chemiluminescent species) would be added to the sample either with thecoated conducting particle, before or after. This mixture of coatedconducting particles, sample and labelled antibodies and/or antigens wouldthen be allowed to bind. This would result in the formation of antibody-antigencomplexes on the surfaces of the various sized conducting particlesand thus the immobilisation of the labels attached to the antibodies and/orantigens in amounts related to the amounts of the analytes in the sample.At this point, the sample may be analysed with or without addition offurther buffers. Further buffers may be needed to assist in the optimalflow by dilution, and/or electrochemiluminescence and/orchemiluminescence of the coated conductive particles in the appliedvoltage field, This mixture would then be aspirated into the flow cell ofthe instrument as outlined above in this example. As it flows through thenarrow portion of the flow cell, the sample will generate light from thevarious sized particles at specific positions in the flow cell path depending on the size of the particle. The location of the light is determined by theuse of light detectors positioned such that light is detected at definedpositions in the flow path. This detection of light can be achievedadvantageously by the use of a diode array or similar as described earlier.In addition, the light emitted may be subjected to spectral analysis.
This flow-through assay system based on conducting particles thusoffers great value as it provides multiple analyte detection and/orquantitation using particle size. This advantage of the flow-through systemis enhanced by its ability to carry out multi-analyte detection usingmultiple wavelengths. Here, each particles light emission is analysedspectrally, and a spectral signature is all that is required to identify andthus assay a given particle. Since each particle is spectrally analysed as itpasses the detectors, labels with overlapping spectra are readily detectableusing, for example, in its simplest form, a 2 spectral window detectionsystem. Such a system based on two spectral windows can readily detectmultiple labels with minor differences in peak emissions when the labelsare excited and analysed separately. Such a system has been used to detectthe presence of at least 4 labels in a DNA sequencing system produced byDuPont. In the case of the system used by Luminex 64, different beads aredetected based on two fluorescent dyes, one orange and one red.
An alternative cell configuration consists of electrodes which flankthe narrow portion of the flow cell with a voltage applied across the pathof the particle as it flows through the cell.
In an additional embodiment of the flow-through system describedabove, detection of the particle may also be incorporated into theinstrument. A number of methods could be used based on methodscurrently employed for particle analysis or cell analysis. An example ofthis is the system used in the copalis technology (Sienna Biotech,Columbia MD) where size is determined by light scattering. Examples ofsuch methods which could be advantageously incorporated in the flow-throughsystem are given hereafter. Light or advantageously a laser could be used to detect and analyse the particles as they pass through thedetection zone. This could be enhanced by the use of coloured orfluorescent structures of conducting and/or semi-conducting material ofthe subject invention to enable additional particle identification formultiple analyte detection in addition to the use of particle size and ECLwavelength. Such a system is used by Luminex(http://www.luminexcorp.com; Fulton et al Clin Chem, 1997, 43, 1749)where two fluorescent dyes are used to code beads into 64 different groupsand a single green fluorescent dye is used for the various assays. The flow-throughsystem of the subject invention could also make use of thismethod for particle identification but may also make use of anelectrochemically detectable species, advantageously an ECL label. Theaddition of a light source to the flow-through instrument may not requirea supplementary detector but could make use of the existing detector usedfor the ECL emission. In order to detect the ECL with the light from thelight source, this light source could be pulsed at a given frequency suchthat the ECL and fluorescent light emission could be multiplexed and thendiscriminated by the light detector and analysis system. The advantage ofthis system is that it would provide information concerning the size andspectral properties of the structure of conducting and/or semi-conductingmaterial of the subject invention and correlate this with the ECL for theparticle. Thus, this system would allow for multiple analyte detectionand/or quantitation. Based on results obtained with the Luminex system,64 to 500 assays may be readily carried out with this type of system.
Alternatively, the particles may be detected and analysed as theyflow through the neck or narrow zone by analysis of the resistance acrossthe neck. This method has been widely used for particle and cell analysisinvented by Coulter and is often called the aperture impedance orelectrical method.
In an alternative embodiment of the subject invention, structures ofthe subject invention coupled to binding species may be used to bind to cells resulting in aggregation or the formation of multiple beads around acell or multi-cell collection such as an embryo or micro-organism. Theseaggregates of more than one bead would then be detected based on theintensity of the light emitted as they pass through the elevated voltagezone which activates the electrochemistry on the surface of the structures.Advantageously, this analysis would make use of the structures as labelsand most advantageously the detectable electrochemistry would be ECL.Thus, after binding the structures, the cells would be added to a buffercontaining an ECL species and a coreactant if needed to allow ECL at thesurface of the structures as they pass the activation and detection zone.Advantageously, the structures used in this embodiment would be beads,particles, nanotubes or fibrils. More advantageously, the structures wouldbe nanotubes or fibrils. Most advantageously, the structures would begraphitic carbon nanotubes or fibrils.
In an alternative embodiment of the flow-through cell configurationof the subject invention, analytes may be detected for eluates from gels,HPLC, FPLC, chromatography and electrophoretic separation methods. Inthis embodiment, a flowing stream from the separation method would bemixed with a flowing stream of buffer, containing structures of the subjectinvention.
This mixture would then flow through a cell as described in theabove embodiments. This would result in the activation of the structuresof the subject invention to carry out electrochemistry.
The results of the activation of electrochemistry would generate adetectable signal. An example could be the detection of amines fromHPLC as has been described (Forbes et al 1997, Analytica Chimica Acta347, 289-293) using conventional electrochemical methods. For thedetection of amines from an HPLC eluate, a flowing stream of Ru(bpy)32+with the structures of the subject invention would be mixed with theHPLC eluate and then passed through a cell where the voltage gradient is applied as described above. Advantageously, the structures would becarbon fibres or nanotubes.
It will be understood that this mode of detection can be applied toany detectable electrochemical reaction by judicious choice of the knownbuffers and coreactants.
In general, the methods used to construct flow cells able to create aflow of individual particles through a zone or neck in the flow are wellunderstood. Thus, the construction of variations which draw on thecurrent knowledge for particle and cell analysers is understood.
In a further embodiment of the subject invention, an alternative useof the structures of conducting and/or semi conducting materials as labelsattached to a binding species is considered. In this example,advantageously, the structure is a carbon nanotube or fibril(advantageously 0.02 to 50 µm, most advantageously 0.5 to 5 µm in length)which is advantageously labelled with a binding species such as anantibody, nucleic acid, or receptor ligand. This binding species-attachedcarbon nanotube or fibril is then used as a reagent in binding to samples ofbinding species present on a surface. This may be the surface of a syntheticmaterial but advantageously the surface is a biological material grown ordeposited onto a surface such as a glass microscope slide. Examples of suchare tissue sections, cell cultures, immobilised arrays of nucleic acid, smearsof tissue which are processed to preserve the biological material ofinterest, stabilise the biological material and improve the presentation ofthe biological material. After specific binding of the binding species on thecarbon nanotube or fibril to the biological sample, the surface is washedthoroughly and placed in the central zone of a cell which contains ECLreagents that may be in organic solvents. Advantageously, the ECLreagents are either tripropylamine and Ru(bpy)32+ or hydrogen peroxideand luminol or luminol, hydrogen peroxide and ferrocene. This cell,which contains the two externally connectable electrodes flanking the central zone, is place in an instrument as described earlier for theapplication of the external voltage and detection of the ECL lightemission. In this case, the use of a microscope and CCD camera has certainadvantages in the instrument in that it provides a spatial determination ofthe position of the bound carbon nanotubes or fibrils on the surface of thesample. This use of the subject invention allows for sensitive detection ofbinding species on a surface and also provide the potential to determinespatial information concerning the distribution of a given binding specieson a cell or within a cell, or the counting of the given cell type within apopulation.
The assays described are directed at the detection of bindingreactions, or the detection of a soluble analyte directly or generated by achemical or enzymatic reaction. It will also be understood that otherchemical and enzymatic assays may also be carried out using a number ofthe formats outlined above. These chemical and enzymatic assays would bebased on both synthesis reactions and degradation reactions.
For example, a synthesis reaction would be the action of DNApolymerase on a template nucleic acid initiated by a primer. In thisexample, the primer could be labelled with an electrochemically detectablespecies such as an ECL label and during the polymerase reaction a bindingspecies could be incorporated such as biotin using a biotinylated NTP. Theresult of such a polymerase reaction would be a nucleic acid strandcontaining both a binding species and an electrochemically detectablespecies. This nucleic acid strand could then be captured onto a structure ofthe subject invention and analysed to determine the level of polymeraseactivity. Examples of synthetic enzymes are DNA, RNA polymerase,glyco-transferases, terminal transferases.
In the case of a degradation reaction, for example a protease or otherhydrolase, the substrate could be coupled to the structure of the subjectinvention in such a way that it contains an electrochemically detectable species which is removable by the action of the specific protease orhydrolase. These modified structures are then incubated with the proteaseor specific hydrolase. Incubation in the presence of an enzyme able tohydrolyse the substrate, and thus remove the electrochemically detectablespecies, results in a loss of signal. Examples of hydrolases are glycosidases,nucleases, protease and lipases which may be used in assays as above.
The following non-limiting examples are given by way ofillustrations and are not to be considered a limitation of this invention,many apparent variations of which are possible without departing fromthe spirit or scope thereof. It will be understood that either the enzyme orthe substrate may be assayed using the methods outlined above.
Example of ECL apparatus and a method for obtaining a detectablesignal from electrolyte-suspended conducting particles.
Basically, the apparatus (illustrated in schematic no. 1 below)integrates a luminometer, a voltage control circuit and a cell with twoexternally electronically connected electrodes.
In schematic no. 1 above: 1 is a potentiostat (voltage generator), 2 isa platinum electrode, 3 is a buffer, 4 is the narrow cross section window ofthe cell, 5 is a luminometer, 6 is a photomultiplier or photodiode, 7 is acontrolling computer.
Optionally, the apparatus may have a pump or other fluid-handlingcomponents and a flow-through cell with externally electronicallyconnected electrodes.
The ECL apparatus consists of a cell with two electrodes at each endand a centre section which is visible to a photomultiplier tube (PMT)(Hamamatsu) powered by a Pacific Instruments Photometer. The data fromthis photometer is digitised and analysed by a computer (Dell Corporation XPS 200, software: National Instruments, Austin Tx). The voltage appliedto the two external electrodes (from a Spellman SL series high voltagegenerator) is controlled by the computer in such a way that the voltageapplied to the centre section of the cell during a measurement cycle iscorrelated with the light level detected at the PMT. A voltage gradient ofup to 35 kv/cm is applied across the central section of the cell whichcontains the sample to be measured. For safety reasons, the voltage controlis constructed to prevent arcing with high current. The centre section ofthe cell, visible to the PMT, is designed with a narrow cross section suchthat most of the voltage gradient applied across the two externallyelectronically connected electrodes is present in this section. The sectionsof the cell which contain the externally electronically connected electrodesare designed such that they are not visible to the PMT during ameasurement. This instrument is designed for multiple cell configurationsto be used:
One cell has a continuous channel from one end to the other whichallows samples to be introduced using a pump or other flow controlmethod. In this case, the two externally electronically connectedelectrodes are attached to the cell via a porous frit, permeable or semi-permeablediaphragms or membranes such that electrical contact is madethrough an electrolyte to the centre section which forms the measuringzone. This cell configuration allows samples to be placed in the measuringzone and then excited via the applied electric field between the twoexternally electronically connected electrodes. The light emitted isdetected and the sample then flushed from the system followed by cleaningand introduction of the next sample for analysis. This cell configurationhas windows in the measuring zone which would also permit fluorescenceand spectrophotometric measurements to be made.
The second cell is configured as in the first cell with respect to theexternally electronically connected electrodes but does not have acontinuous channel from one side to the other. Instead, the sample isintroduced via a port in the centre section of the cell. The externallyelectronically connected electrodes are isolated from this centre section viaa porous frit or semi-permeable membrane. This cell allow manual washingand introduction of the sample followed by a measurement cycle.
The third cell is designed as for the second cell but the centresection is modified such that it is able to accept or hold a filter paper orimmunochromatographic filter element or gel. This centre section is ableto accept the filter paper or immunochromatographic filter element or gelin such a way that electrolyte is in contact with the filter paper orimmunochromatographic filter element or gel and the externallyelectronically connected electrodes. This centre section with the filterelement also has a window to hold the filter element in place and to allowclose proximity of the PMT to the filter element, improving photoncapture by the PMT. The window element is variously glass, plastic, andplastic wrap which is transparent to the photon wavelengths of interest.
The above instrument is also configured to allow the use of aphotodiode in place of the PMT.
To test the above ECL apparatus and cells (cell 2), a suspension ofglassy carbon spheres 20-50 µm (#41260, Alfa Aesar) at 20-200µg/ml inECL buffer (200 mM potassium phosphate, 100 mM tripropylamine, 0.02%(w/v) Tween 20, pH 7.2) with 1nM Ru (2,2-bipyridyl)32+ [Ru (bpy)32+] isintroduced manually intocell 2 through the port in the centre section ofthe cell. The externally electronically connected electrode chambers arefilled with ECL buffer. The cell is then placed in proximity to the PMTsuch that it can substantially see the centre section of the cell but not thechambers which contain the externally electronically connected electrodes.The PMT and cell combination is placed in a dark environment to allow the specific detection of the light emitted from the ECL at the surface ofthe glassy carbon spheres. The results of this experiment demonstrate apeak of detectable light which is dependent on the level of the appliedvoltage. In this case, light is seen when a voltage of up to 3 kV/cm isapplied in the form of a ramp of increasing voltage from 0 to 3 kV/cm.The ECL signal is found to increase with increasing numbers of particlesand with increasing concentrations of Ru(bpy)32+.
Carbon materials (particles, fibres, nanotubes, fibrils) are obtainedfrom Alfa Aesar (Ward Hill, MA) and MER corporation. These carbonmaterials are made into an aqueous slurry by mixing 0.1% w/w withdeionized water and dispersing by sonication using a 400 watt sonicationprobe for 0.5 to 1 hour. These carbon materials are then ready forchemical activation by oxidation using chromic acid, perchioric acid(Dong et al 1996, J Mol Rec. 9, 383-388) (to introduce carboxyl groups) orfor direct coating with biomolecules. Carbon fibres and various carbonparticles and other such structures are obtained from Alfa Aesar (WardHill, MA). This oxidation is achieved following standard protocols asfollows. For example, 10 g of the carbon fibrils, particles, fibres or otherstructures are mixed with 450 ml of conc. H2SO4. This preparation is thenmixed with 8.68 g of NaClO3 under argon by adding small amounts over a24 hr period. The mixture is then added to ice and followed by extensivewashing. The final carboxylated structures are dried and a portion used todetermine the carboxylic acid content by titration of these groups. Thechromic acid protocol is similar except that the reaction is not conductedunder argon.
Carbon material suspensions as described in example 1 are added toPBS to generate a 0.1-1 mg/ml suspension of carbon. These are washedonce then antibody or streptavidin is added at a concentration of 100-300µg/mlin a pH 9.6 100 mM sodium carbonate/bicarbonate buffer. Thefibril mix is left to mix at 48°C for 16-20 hours. Following thisincubation, the fibrils are washed by centrifugation and resuspended in 3%BSA, 0.1% Tween 20 PBS and incubated for 2 hours to block any uncoatedsites. These fibrils are then washed 5 times in the 3% BSA, PBS with 0.05%sodium azide and finally resuspended in this buffer for storage. Prior touse, the fibrils are washed into PBS 0.1% Tween 20 to a concentration of0.1-1 mg/ml.
In the case of other carbon particles, these are prepared by a quickwash in PBS followed by addition of the antibody-containing solution andapplication of the protocol above.
Carbon particles, fibres and fibrils are obtained from MERcorporation and Alfa Aesar (Ward Hill, MA). These are activated usingoxidative methods such as perchloric acid, chromic acid treatment, oxygenplasma or other methods known in the art to generate -COOH functionalgroups on the surface of carbon, glassy carbon and graphitic carbon (seeexample 1). These carbon surfaces - derivatized with COOH groups - aresuspended in anhydrous dioxane with mixing to concentrations of between0.1 and 10 mg/ml. An estimate of the moles of COOH groups per mg ofparticles is made by titrating the COOH groups on a sample with NaOH.To this mixture is added a 5-30 fold molar excess of N-hydroxysuccinimidefollowed by mixing to obtain dissolution. When the N-hydroxysuccinimidehas dissolved, an equivalent molar amount of ethyl-diamino-propyl-carbodiimide (EDAC) is added and the mixture incubatedwith mixing for 1-4 hours at room temperature (20-24°C). Following thisincubation, the particles are separated from the mixture either byfiltration (particles larger than 20µm) or by centrifugation. The particlesare washed with anhydrous dioxane several times followed by ananhydrous methanol wash. These resultant NHS ester-activated particlesare then dried under vacuum and stored under anhydrous conditions untilused for coupling
For coupling, between 10 and 1 mg of dried NHS-ester particles arewashed with PBS and added to a solution of the biomolecules to becoupled, typically streptavidin, antibody and amino-modified DNA oroligonucleotide. In the case of streptavidin, a 5-10 mg/ml solution in PBSis used. For antibody, between 0.5 and 1 mg/ml of antibody is used tocouple to 1-3 mg of particles. For amino-modified oligonucleotides,between 0.1 and 1 mg/ml of oligonucleotide between 15 and 40 bases longis used. The particles and biomolecules are mixed to form a suspension andthis is then incubated for 1-2 hours at room temperature. The couplingreaction between the amino groups on the biomolecules and the NHS-estergroups on the particles forms an amide bond between the biomolecule andthe particle. These particles are then resuspended in 3% BSA, 0.1% Tween20, PBS and incubated for 2 hrs. They are then washed and resuspended inthe same buffer.
1 mg of protein is labelled with Ru (2,2-bipyridyl))32+ (Ru(bpy)32+).The protein is buffer-exchanged using Centricon 30 microconcentrators(Amicon) into 0.15M potassium phosphate buffer, 0.15M NaCl pH 7.8, thefinal volume being 0.5 mL. Immediately prior to use, 0.5 mg ofRu(bpy)32+-NHS (Ru(2,2-bipyridyl)2(4-[3-(1,3-dioxolan-2-yl)propyl]-4-methyl-2,2-bipyridine)2+)is dissolved with 125µl of anhydrous dimethylsulfoxide (Aldrich). A molar ratio of about 25:1 should be achieved between the Ru(bpy)32+ and the protein, based on molecular weights of1057 for Ru(bpy)32+-NHS and 150,000 for antibodies. The Ru(bpy)32+-NHS(45µl) is added to the protein solution while shaking. The ranges forlabelling can be from 3:1 to 30:1 molar ratios depending on the proteinbeing labelled. The reaction tube is incubated in the dark at roomtemperature for 30 minutes while shaking. The reaction is terminated bythe addition of 25µl of 1M glycine and incubation for 10 minutes. Thereaction mixture is purified by passage through a Sephadex G-25 column (1X 20 cm in 0.15M potassium phosphate, 0.15M NaCl with 0.05% sodiumazide pH 7.2). The Ru(bpy)32+-labelled protein fractions are collected andpooled. These labelled proteins are then diluted into PBS buffer with azideto give protein concentrations between 1.6µg/ml and 32µg/ml.
Immunoassay for the quantitative determination of human chorionicgonadotropin. This electrochemiluminescent assay demonstrates theprinciples of the subject invention.
The measurement of HCG concentrations can be used to diagnosepregnancy just one week after conception.
Reagents are obtained from Boehringer Mannheim, i.e. HCG STATimmunoassay reagents (Cat #1731 289) sufficient for 100 tests. This test kitis used to provide the two antibodies, one being labelled with anelectrochemically detectable species (Ru(bpy)32+), the other being abifunctional binding species composed of an antibody conjugated tobiotin. The solid phase corresponds to the glassy carbon beads prepared inexamples 3 and 4 and coated with streptavidin. In addition, the HCGSTAT calibrator set (Cat # 1731670), the buffer to provide optimal ECL,i.e. ProCell (Cat # 1662988) (this buffer is equivalent to the ECL bufferused in example 1 and to ORIGEN assay buffer) and Elecsys DiluentUniversal (Cat # 1732277) are obtained from Boehringer Mannheim.
The samples are prepared from the HCG STAT calibrator set bydilution in Elecsys Diluent Universal. One of the calibrators in the HCGSTAT calibrator kit consists of about 10 mIU/ml and the other about 5000mIU/ml. The highest calibrator is used to make a series of samples byserial dilution in the Elecsys Diluent Universal to give a sequence of HCGvalues which form the basis of the demonstration. The result of thedilutions is a series of HCG values: 5000 (from the original calibrator),1250, 625, 156, 78 and 20 mIU/ml.
The assay is performed as follows; 40 µl of the diluted calibratordescribed above is added to 220 µl of the biotinylated antibody and 220 µlof the Ru(bpy)32+-labelled antibody. This is mixed and incubated for 5-10min followed by addition of 100µg (100 µl) of streptavidin-coated 2-4µmglassy carbon particles as prepared in examples 3 and 4. This mixture isincubated for 5-10 min with mixing. This mixture of assay components isthen centrifuged and resuspended in the ProCell buffer and washed againbefore final resuspension in 600 µl of the ProCell buffer. These samples inthe ProCell buffer are then introduced into the cell of the instrument asdescribed in example 1 and the light given off for each sample isdetermined. The values are background-subtracted using the 0 HCG levelsample and normalised to a value of 1000 for the 5000 mIU/ml calibratorsample. The results demonstrate an increase in light detected withincreasing HCG levels in the samples.
Immunoassay for the quantitative determination of oestradiol. Thiselectrochemiluminescent assay demonstrates the principles of the subjectinvention in a competitive assay format.
Measuring oestradiol concentrations is important in thedetermination of fertility disorders derived from the hypothalamus-pituitary-gonadaxis, gynecomastia and tumours.
Reagents are obtained from Boehringer Mannheim Oestradiolimmunoassay reagents (Cat #1776002) which contains reagents for 100tests. This test kit is used to provide the two binding species for the assay:oestradiol with a peptide linker labelled with an electrochemically-detectablespecies (Ru(bpy)32+), an antibody used as a bifunctional bindingspecies and another antibody conjugated to biotin. The solid phasecorresponds to the glassy carbon beads prepared in examples 3 and 4 andcoated with streptavidin. The Oestradiol CalSet (Cat # 1776037), thebuffer to provide optimal ECL, ProCell (Cat # 1662988) and ElecsysDiluent Universal (Cat # 1732277) are obtained from BoehringerMannheim.
The test samples are prepared from Sigma reference standardoestradiol (Cat # E 1132) made up in Elecsys Diluent Universal. Inaddition, test samples from the Oestradiol CalSet (Cat # 1776037) are runin some experiments.
The assay is performed as follows; 100 µl of the sample are added to180 µl of the biotinylated polyclonal anti-oestradiol antibody from the kitand incubated for 10-15 min followed by addition of 180 µl of theoestradiol-peptide linker-(Ru(bpy)32+) and 100 µl of streptavidin-coatedglassy carbon particles (100µg of 2-4µm coated glassy carbon particlesprepared as in examples 3 and 4). This mixture is incubated for 10-15 minwith mixing. This mixture of assay components is then centrifuged andresuspended in the ProCell buffer and washed again before finalresuspension in 600 µl of the ProCell buffer. These samples in the ProCellbuffer are then introduced into the cell of the instrument as described inexample 1 and the light given off for each sample is determined. Thevalues are background-subtracted using a sample not containing anyoestradiol. The results demonstrate an increase in light detected withincreasing oestradiol levels in the samples.
100µl serum calibrators for TSH, 25µl of ECL-labelled mouse anti-TSHprepared as in example 5 (typically the amount of antibody added toa test is between 40 ng and 800 ng) and 25µl (between 3 and 100µg) ofglassy carbon structures (1-10µm) coated with sheep anti-TSH in ECLbuffer following the protocols given in examples 3 and 4, are combinedand incubated in polypropylene tubes for 15 minutes at room temperaturewith mixing. The glassy carbon structures are then washed twice bycentrifugation and resuspended in ECL buffer to a final volume of 1 ml.These samples are then analysed for ECL using the instrument as describedin example 1. The ECL signals from the various samples are then plottedagainst the serum calibrator values to generate a standard curve. Thisdemonstrates that the method allows the determination of analytes usingsimple binding interactions as seen in antibody-antigen, receptor-ligandand nucleic acid-nucleic acid binding reactions. This standard curve canthen be used to determine the level of unknown samples of the TSHanalyte.
This assay was repeated without washing the glassy carbonstructures and demonstrated the use of this format for a no-wash, one-stepassay without particle separations and/or washings.
Oligonucleotides are obtained from Oligo etc (Wilsonville, OR,USA). Amino-modified oligonucleotides are labelled with Ru (2,2-bipyridyl)32+using the NHS ester or made directly during synthesis usingRu (2,2-bipyridyl)32+ phosphoramidite obtained from IGEN Inc.
Beta-actin PCR, nucleic acid probe assay.
The PCR detection of beta-actin mRNA is achieved by theconversion of mRNA into cDNA followed by a PCR which includes abiotinylated primer and an ECL labelled primer. In this protocol, the ECLlabel used is ORIGEN TAG (IGEN, Gaithersburg MD). The followingprimers are used to amplify the beta-actin cDNA by PCR; 5' Biotin-GCC ACA GGA TTC CAT ACC CAA-3' and 5'ORIGEN TAG-GAG AAGAGC TAT GAG CTG CCT GAC-3'.
From 0.1 to 5 micrograms of mouse liver total RNA, includingcontrols not containing any beta-actin, are reverse-transcribed in a 20µlreaction for 1 hr at 42°C with Superscript II RNase H- reversetranscriptase, oligo dT, and 10mM dNTP mix as recommended by themanufacturer (Life Technologies) to generate the cDNA. One microliter ofcDNA is amplified in a 100 µl reaction containing 1XPCR buffer (PerkinElmer), 200 µM dNTPs (LTI), 20 pmoles of each primer, and 4 units ofAmplitaq DNA polymerase (PE) pre-mixed 1:1 with TaqStart Antibody(Clontech). Amplification is performed on a Perkin Elmer Model 480Thermal Cycler using the following conditions: 10 minutes at 50°C; 10minutes at 94°C; 20 cycles of 45 seconds at 94°C, 45 seconds at 64°C, 2minutes at 72°C followed by a final extension of 7 minutes at 72°C.Following the PCR, 3 µl of the reaction mixture are added to 2-40 µg ofstreptavidin-coated glassy carbon structures (as prepared in examples 3 and4) in 50µl of ECL buffer. This mix of PCR products and the streptavidin-coatedglassy carbon structures is mixed for 30 minutes at roomtemperature followed by the addition of 250 µl of ECL buffer. Followingthis dilution step, the glassy carbon structures are washed in ECL bufferand then placed into the central section of the ECL instrument describedin example 1 for ECL excitation and detection. The results demonstrate anECL signal dependent on the level of the beta-actin DNA in the samples.
Solutions containing cholesterol (30µl of 2-50µM), cholesterol oxidase(10µl of 48 units/ml) and buffer (120µl of 100 mM NaPhosphate, pH5containing 0.05% Triton X-100) are prepared and incubated for 30 min. Themixture is then rendered 0.2 mM with respect to luminol using a solution ofluminol in 0.05M sodium borate buffer pH 10.0. In an alternative assayformat, following the 30 min incubation step, the mixture is rendered 100µM ferrocene, 100µM luminol and 0.1 M Tris buffer. To these mixturesare added 2-80µg of glassy carbon particles (Alfa). The samples are thenplaced into the ECL instrument described in example 1 followed byexcitation and detection of the ECL light. The samples show an increasingsignal with increasing cholesterol, forming the basis of a cholesterol assay.Stock solutions of cholesterol are prepared by dissolving cholesterol in amixture of Triton X100 and ethanol (3:2 v/v). The particles used in thisassay are untreated 0.2-5µm glass carbon beads.
The basic reagent mix for the glucose assay contains 16.8 µg/mlhexokinase, 20 µg/ml glucose-6-phosphate dehydrogenase, 1 mM ATP, 1mM NAD+, 2 mM MgSO4 and 25 mM Tris HCL, pH 7.5. Aliquots of thisreagent mix are prepared (270µl) and 30µl of various glucose standards areadded to give a final concentration range of 5 to 35µM. These samples areincubated for 10 min at room temperature. Following this incubation, 30µl of 10mM Ru(bpy)32+ is added followed by 2-40µg of glassy carbonparticles (Alfa). The samples are then placed into the ECL instrumentdescribed in example 1 followed by excitation and detection of the ECLlight. The samples show an increasing signal with increasing glucose,forming the basis of a glucose assay. The particles used in this assay areuntreated 0.4-12µm glass carbon beads.
A solution of benzylpenicillin (1 mM) is prepared in 0.1 Mphosphate (sodium salt), 0.05% Triton X-100 pH 7.5. This solution isrendered 10µm with respect to ruthenium (II) tris (bipyridyl)(Ru(bpy)32+). This mixture is used as the buffer for the detection ofbetalactamases. To 1 ml aliquots of this mixture are added E. coli betalactamase RTEM to a concentration of between 1 and 10 nM. The enzymereaction is allowed to proceed for about 10 min at room temperature (about 20°C). After the enzyme incubation, 2-40µg of glassy carbonparticles (Alfa) are added and the mixture is placed in the central zone ofthe cell of the instrument described in example 1. The sample is thensubjected to an applied voltage and the ECL signal is measured. It is foundthat ECL signal is significantly higher in the presence of the beta-lactamaseenzyme. This allows a method to be developed for the detectionof beta-lactams and betalactamases. The particles used in this assay areuntreated 0.4-12µm glass carbon beads.
Carboxylate-modified carbon structures prepared as in example 2,(2.5µg) are added to 1.0 millilitre (ml) of methyl ethyl sulfonate (MES)buffer (5 millimolar (mM), pH 4.75) and 75 microliters of antibodysolution (antibody to beta-hCG, Biogenesis UK) (2 milligrams permillilitre (mg/ml)). The solution is stirred and 100 ml of 1-Ethyl 3(3-Dimethyl-aminopropyl)carbodimide HCl (EDAC) (2 mg per 10 ml H2O)are added. The solution is stirred overnight at 2-8°C, after which thestructures are isolated by centrifugation, washed twice with 0.1% "Tween-20"solution, and resuspended in "PBS" Phosphate Buffered Saline (0.01 MKH2PO4; 0.15 M NaCl: pH 7.2) to yield a 0.125% solution. Afterresuspension in PBS, the structures are stored at 2-8°C for subsequent usein the following procedures.
50 microliters of the antibody-coated structures from Example 13 areadded dropwise to the centre of a Whatman GF/D glass filter; 100microliters of pig sera are then added and the filter and microparticlesincubated for 30 minutes in a humidity chamber at room temperature.After this time, the filter - now containing the microparticles - is washedthree times in 300 microliters of PBS buffer. The filter is then stored in ahumidity chamber for use in the following immunoassay example. The microparticles are observed, by scanning electron microscopy, to havebeen trapped or agglomerated on the glass fibres of the filter material.
It should be noted that, in addition to the techniques described inthe foregoing example, antibody, antigen, ligand, receptor and nucleic acidbinding sequences may be attached to the particles by a variety ofmethods, e.g., adsorption or use of various chemical activators. Also, it isto be appreciated that the particles can be added to the fibrous matrixafter, for example, animal sera or other blocking agents have been added,and that the use of such sera is not of critical importance. Therefore, theorder of addition of the particles to the matrix and treatment thereof afteror before incorporation into the matrix is not critical to the presentinvention. Moreover, it will be appreciated that fibrous materials can beused in place of the glass filter matrix material specifically describedherein, and the advantages of the invention can also be realised thereby.
The glass fibre material containing the antibody-coated structures aspreviously described (example 14), is cut into substantially circular"disks", and the disks - forming reaction matrices - are placed in contactwith a blotter material to absorb excess fluid from solutions used in theassay. Thereafter, five drops of test samples of human urine (about 280microliters) containing zero, and 50 and 100 mIU/ml levels of beta-hCG(Table 1, infra) are added to each matrix after passage of the sample dropsthrough a prefilter situated above each matrix. Three drops of an antibodylabelled with an ECL active species, as used in example 6, are then addedto each matrix through the prefilter, and each matrix is incubated at roomtemperature for about two minutes. The prefilter is then removed and 1.0ml of PBS, 0.1% Tween 20 wash solution is added to each matrix toremove any excess antibody-enzyme conjugate. One drop of ProCell bufferis then added to each matrix. After two minutes, each matrix is placed in a cell (example 1, cell 3) containing two externally and electronicallyconnected electrodes (see above description of ECL instruments). Thematrix placed in the cell forms an electrical contact between the externallyconnected electrodes via the ProCell buffer which forms the electrolyte.
Each matrix is then subjected to an externally applied voltage. Thisvoltage is applied at such a voltage gradient up to 10 kv/cm that at leastsome of the structures trapped in the glass fibre filter are rendered bipolar.These bipolar structures activate the ECL from the captured antibodylabelled with an ECL active species and light is detected for the testsamples which contains beta-hCG. The amount of light is correlated to thelevel of beta-HCG in the sample.
Glassy carbon particles are purified using sucrose gradientcentrifugation to isolate particles within a range of 0.2 to 2µm indiameter. These particles are then activated using the oxidation reactionsdescribed above and activated to form the NHS ester as described. Thesebeads are then reacted with BSA biotin from Pierce (Rockford, IL, cat #29130). The resultant particles are then reacted with excess BSA to blockany remaining binding sites and the beads are washed 5 times over a periodof 3 days. Large pore nitrocellulose FF85 from Schleicher and Schuell(Ecquevilly, France) is cut intostrips 1 cm by 10 cm and a strip ofstreptavidin applied 3 cm from one end of the nitrocellulose membrane.These membranes are then washed in water and incubated in 3% BSA inPBS overnight followed by washing in water and air-dried. Thesemembranes are then placed into tubes containing 0.3 ml of the abovebiotinylated glassy carbon particles with and without added biotin andallowed to stand for 2 hrs such that the glassy carbon particles may wickup the dry membrane. The membranes are then washed with a solution of0.1 mM luminol, 1 mM hydrogen peroxide, 0.1 mM ferrocenemonocarboxylic acid (Sigma) and 0.05M Tris buffer pH 8. The membranes are then trimmed to remove the lower and upper portions of the strip toleave the site of streptavidin strip. The streptavidin strip portion is placedinto a cell designed to provide electrolyte contact to two externallyelectronically connected electrodes attached to a voltage control circuitcontained within the instrument as described in example 1. Thesemembranes are then subjected to the voltage field and the ECL is detected.The strips run in the absence of added biotin show higher ECL signalsthan those run in the presence of free biotin which blocks the binding ofthe biotinylated glassy carbon particles and prevents the binding of theseto the immobilised streptavidin on the nitrocellulose membrane. Thebinding of the glassy carbon particles immobilises a conductive structureof the subject invention which is then able to activate ECL when anexternal voltage is applied. This therefore demonstrates the potential ofthis system to form the basis of a system used to carry out specific bindingreactions with enhanced sensitivity.
